Transgenic plants used as a bioreactor system

ABSTRACT

The present invention relates generally to the use of plants as bioreactors for the production of molecules having useful properties such as inter alia polymers, metabolites, proteins, pharmaceuticals and nutraceuticals. More particularly, the present invention contemplates the use of grasses, and even more particularly C4 grasses, such as sugarcane, for the production of a range of compounds such as, for example, polyhydroxyalkanoates, pHBA, vanillin, indigo, adipic acid, 2-phenylethanol, 1,3-propanediol, sorbitol, fructan polymers and lactic acid as well as other products including, inter alia, other plastics, silks, carbohydrates, therapeutic and nutraceutic proteins and antibodies. The present invention further extends to transgenic plants and, in particular, transgenic C4 grass plants, capable of producing the compounds noted above and other products, and to methods for generating such plants. The ability to utilize the high growth rate and efficient carbon fixation of C4 grasses is advantageous, in that it obviates the significant growth penalties observed in other plants, and results in high yields of desired product without necessarily causing concomitant deleterious effects on individual plants. In addition, the C4 grass, sugarcane, is particularly advantageous, as in addition to the features common to all C4 grasses, this plant accumulates sucrose. This sucrose store provides a ready supply of carbon based compounds and energy which may further obviate any deleterious effects on the growth of the plant associated with the production of the product. The present invention provides, therefore, a bioreactor system comprising a genetically modified plant designed to produce particular metabolic or biosynthetic products of interest.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the use of plants asbioreactors for the production of molecules having useful propertiessuch as inter alia polymers, metabolites, proteins, pharmaceuticals andnutraceuticals. More particularly, the present invention contemplatesthe use of grasses, and even more particularly C4 grasses, such assugarcane, for the production of a range of compounds such as, forexample, polyhydroxyalkanoates, pHBA, vanillin, indigo, adipic acid,2-phenylethanol, 1,3-propanediol, sorbitol, fructan polymers and lacticacid as well as other products including, inter alia, other plastics,silks, carbohydrates, therapeutic and nutraceutic proteins andantibodies. The present invention further extends to transgenic plantsand, in particular, transgenic C4 grass plants, capable of producing thecompounds noted above and other products, and to methods for generatingsuch plants. The ability to utilize the high growth rate and efficientcarbon fixation of C4 grasses is advantageous, in that it obviates thesignificant growth penalties observed in other plants, and results inhigh yields of desired product without necessarily causing concomitantdeleterious effects on individual plants. In addition, the C4 grass,sugarcane, is particularly advantageous, as in addition to the featurescommon to all C4 grasses, this plant accumulates sucrose. This sucrosestore provides a ready supply of carbon based compounds and energy whichmay further obviate any deleterious effects on the growth of the plantassociated with the production of the product. The present inventionprovides, therefore, a bioreactor system comprising a geneticallymodified plant designed to produce particular metabolic or biosyntheticproducts of interest.

2. Description of the Prior Art

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Modern techniques of biotechnology are driving a new revolution thatpromises both scientific and financial gains for a range of industries.One difficulty, however, is the large financial cost of establishingsufficient infrastructure to generate recombinant products or togenerate the products resulting from recombinant processes. Alternative,more cost effective systems are required to assist the generation oflarge amounts of product resulting from recombinant processes.

For agricultural industries, the generation of genetically engineeredplants enables plants to be quickly developed with desired traits suchas resistance to pathogen infestation. However, plants can also be usedto produce a wide range of compounds not normally produced within theplant, thereby providing a source of renewable raw materials for themanufacturing, energy and pharmaceutical industries.

This endeavour is aided by the fact that plants, animals, insects,bacteria, fungi and even viruses have evolved in a wide range ofdifferent habitats and, hence, produce a remarkable array of compoundswhich allow them to survive and thrive under very varied environmentalconditions. It is estimated that up to 100,000 unique compounds exist inthe plant kingdom alone. In the future, genes and even entire geneticpathways may become available from different sources to assist in themanufacture of a wide range of commercial products.

Traditional chemical industries are increasingly looking towardsbiological systems for the production of bulk and fine chemicals.Biological processes offer numerous advantages over chemical processes,including the elimination of complicated and difficult high pressure andhigh temperature reactions, the use of aqueous systems rather thanorganic solvents, high degrees of product stereo-specificity, a capacityfor highly complex synthesis and comparatively simple scale-up. The useof biological processes is not a new phenomenon, as many fine chemicals(e.g. enzymes, antibiotics) and bulk chemicals (e.g. ethanol, aminoacids, citric acid, lactic acid) are produced effectively in microbialsystems. Advances in molecular biology and genomics have enabled anexpansion of the available product range, the transfer of productionsystems to microbes with desirable production traits, and significantlyincreased yields. Nevertheless, inherent limitations remain, in that theraw materials (e.g. molasses, sucrose, or high fructose corn syrup) andscaled up fermentation processes are relatively expensive.

By contrast, genetically modified plants should not require any more rawmaterials than are already required by their non-transformedcounterparts and have the potential to provide a low cost of productionper tonne of biomass, when compared with fermentation methods. Thus, ifreasonable product yields could be achieved in plants, and if theseproducts could be extracted at reasonable cost, the potential forchemical production in plants would be extremely high.

The polyhydroxyalkanoate (PHA), poly-(D-3-hydroxybutyrate) (PHB), is athermoplastic with physical properties akin to polypropylene. Both PHBand polypropylene are water insoluble, exhibit good gas-barrierproperties and possess similar melting points, degrees of crystallinityand glass-rubber transition temperatures (De Koning, Can. J. Micro.41(1): 303-309, 1995), although PHB is more resistant to UV radiation.Moreover, unlike polypropylene, PHB is rapidly degraded by numerousbacteria and fungi under composting conditions (Jendrossek et al., App.Micro. Biotech. 46: 451-463, 1996; Mergaert and Swings, Indust. Micro.Biotech. 17: 463-469, 1996).

Cost is the major reason why PHA produced by bacterial fermentationcannot compete with conventional plastics production methodologies.Major contributors to the cost of PHA are substrate cost, energyconsumption during fermentation, disposal of waste product, and the costof constructing and maintaining plant and machinery. The use oftransgenic plants for PHA production, however, has the potential toeither eliminate or drastically reduce these costs since atmosphericcarbon dioxide would be the substrate and energy would be derived fromsunlight. Operating costs would be no more than what is incurred inordinary agricultural practices. Waste products are the same as for anon-transgenic crop. This makes plants an attractive potentialalternative to bacterial fermentation.

The production of PHB has been most closely studied in the bacteriumRalstonia eutropha (formerly Alcaligenes eutrophus), which accumulatesPHB at up to 80% of its cell dry-weight (Steinbüchel and Schlegel, Mol.Micro. 5: 535-542, 1991). The PHB biosynthetic pathway within R.eutropha is well known and consists of three steps catalyzed by thethree enzymes 3-ketothiolase, acetoacetyl-CoA reductase and PHBsynthase, respectively. For large-scale industrial production of PHB,bacterial fermentation is economically and environmentally lessfavourable than the corresponding production of petrochemically derivedplastics like polypropylene (Lee, Trends Biotech. 14: 431-438, 1996;Gerngross, Nature Biotech. 17: 541-544, 1999). Hence, since the glucosesupplied to PHB-producing bacteria is derived from plants, it would beadvantageous to be able to produce PHB in plants directly.

Attempts to achieve this goal have been unsuccessful, due largely to thesignificant added burden placed upon individual plants by requiring themto produce this macromolecule, resulting in a severe reduction in plantgrowth and infertility (Poirier et al., Science 256: 520-523, 1992;Bohmert et al., Planta 211: 841-845, 2000).

In accordance with the present invention, an efficient bioreactor systemis developed in plants and in particular Saccharum sp. such assugarcane.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

The present invention provides a plant-derived bioreactor system.Although previous attempts to effect the manufacture of usefulindustrial and other products in plants have not been overly successful,it has been determined in accordance with the present invention thatthis was due to the extra load placed on individual plants whichresulted in deleterious growth effects. C4 grasses, which haveparticularly efficient mechanisms for the assimilation of carbon, areidentified as having a high growth rate and high accumulation of biomassmaking them useful as bioreactors for the production of a wide range ofproducts. Furthermore, the C4 grass, sugarcane, is particularly usefulas this plant stores sugars in dimeric and/or polymeric forms. Thesestores may be utilized when needed such as, for example, for rapidvegetative growth, or for energy during times of significantenvironmental stress. The store of carbohydrate is identified inaccordance with the present invention as providing a ready supply ofprecursor for many metabolic pathways, and utilisation of this storedoes not stress the producing plant. Therefore, the present invention ispredicated, in part, on the identification of a subset of plants, namelythe C4 grasses (particularly sugarcane), as useful bioreactors on thebasis of their high carbon assimilation rate, rapid growth, high biomassproduction and large carbohydrate store, such as is found in the stem.

The present inventors have capitalized on the potential of a crop thathas a highly efficient C4 carbon assimilation mechanism, a rapid growthrate and naturally harbours large quantities of sucrose in its stems,thereby having the ideal properties of a bioreactor. The instantinventors have developed a means to engineer this crop so as toeffectively accumulate significant quantities of a product withoutsignificant decreases in biomass or growth rate. In so doing, the plantscontemplated herein permit the manufacture of products such asbiodegradable plastics, vanillin, indigo, adipic acid, 2-phenylethanol,1,3-propanediol, sorbitol, fructan polymers and lactic acid as well astherapeutic, nutraceutic and diagnostic agents without incurring thepreviously observed deleterious effects on growth and viability.

Accordingly, one aspect of the present invention provides a method forgenerating a plant-based bioreactor system, said method comprisingselecting a plant having a high efficiency carbon assimilationmechanism, rapid growth rate and/or high biomass production and/orreserves of metabolites or having a capacity to generate such reservesand/or which possess metabolic and/or biosynthetic pathways useful inthe manufacture of a product of interest or a precursor form thereof;genetically modifying cells of the plant to enable access to saidmetabolites and/or metabolic or biosynthetic pathways; and thenregenerating a genetically modified plant from said cells.

The present invention is particularly directed to C4 grasses, however,other non-grass C4 plants such as woody or herbaceous plants whichutilise the C4 pathway are also contemplated by, and are within thescope of, the present invention.

In a preferred embodiment of the present invention, the subject plant isa C4 grass. In an even more preferred embodiment the plant is sugarcane.As used herein, the term sugarcane is to be understood to include, interalia, plants of the Saccharum genus, incl. S. robustum, S. offinarum,and S. spontaneum and hybrid Saccharum sp., incl. modern sugarcanecultivars.

The preferred compounds to be produced by the plant bioreactor include:vanillin, sorbitol, polyhydroxyalkanoates (PHA) such aspoly-(D-3-hydroxybutyrate) (PHB), indigo, fructan, lactic acid, adipicacid, 1,3-propanediol, 2-phenylethanol and pHBA. However, the presentinvention also extends to the use of C4 grasses as bioreactors togenerate a compounds such as therapeutics, nutrapharmaceuticals,diagnostic agents including, for example, single chain antibodies,industrial enzymes and the like.

The present invention contemplates, therefore, a method for producing aproduct of interest including a product or intermediate of abiosynthetic or metabolic pathway in a C4 grass, said method comprisingexpressing one or more genetic sequences which encode one or moreenzymes or proteins required for the production of the product orintermediate or a homolog or precursor thereof or which induces genesilencing of genetic material which encodes an enzyme or protein in abiosynthetic or metabolic pathway in cell of a C4 grass plant such thatthe product or intermediate accumulates in the cytosol, storage vacuole,plastid or non-plastid organelles of the cell, or accumulates in thejuice or vascular fluid of the plant.

The present invention therefore provides for the production of a productin a C4 grass wherein product accumulation is at least in partpredicated on the direct activation or inhibition (includingdown-regulation) of an enzyme in a biosynthetic or metabolic pathway bythe administration to the plant of an enzyme inhibitor or activator.Reference to an “enzyme inhibitor or activator” includes geneticmaterials which, for example, induce (post-transcriptional ortranscriptional gene silencing of a structural gene or positive ornegative regulator gene.

In one preferred embodiment, increased accumulation of a product orintermediate from a biosynthetic or metabolic pathway is a result ofinhibition of one or more biosynthetic or metabolic enzymes andoptionally re-directing metabolites down another biosynthetic ormetabolic pathway.

In another preferred embodiment, the present invention contemplates theuse of sugarcane as a bioreactor. Therefore, alteration to the geneexpression profile of sugarcane to effect the production of anendogenous metabolite at an increased level, or to produce anyheterologous metabolite is within the scope of the present invention.Accordingly, induction or supression of any biosythetic genes insugarcane, such as described herein, is to be considered within thescope of the present invention.

Typically, the production of one or more metabolites or heterologousproteins, polypeptides or peptides in a plant is achieved by expressionof a nucleic acid molecule encoding the metabolite or protein,polypeptide or peptide of interest. Any nucleic acid which encodes aprotein, polypeptide or peptide of interest is contemplated by thepresent invention. However, preferred nucleic acids include thoseencoding:

-   -   (i) vanillin biosynthetic enzymes, including 3-dehydroshikimate        dehydratase, catechol-o-methyltransferase, aryl aldehyde        dehdrogenase, feruloyl-CoA synthetase, enoyl-CoA        hydratase/aldolase;    -   (ii) sorbitol biosynthetic enzymes, including glucose/fructose        oxidoreductase;    -   (iii) PHA biosynthetic enzymes, including 3-ketothiolase,        acetoacetyl-CoA reductase, PHA synthase, enoyl hydratase,        3-hydroxyacyl-acyl carrier protein:CoA tranferase;    -   (iv) indigo biosynthetic enzymes, including tryptophanase,        L-tryptophan indole lyase, napthalene dioxygenase, R. eutrophica        bec gene product;    -   (v) fructan biosynthetic enzymes, including        fructosyltransferases and levansucrases;    -   (vi) lactic acid biosynthetic enzymes, including lactate        dehydrogenase;    -   (vii) adipic acid biosynthetic enzymes, including        3-dehydroshikimate dehydratase, protocatechuate decarboxylase        and catechol 1,2-dioxygenase;    -   (viii) petroselinic acid biosynthetic enzymes, including        3-ketoacyl-ACP synthase;    -   (ix) 1,3-propanediol biosynthetic enzymes including glycerol        dehydratase, 1,3-propanediol oxidoreductase,        glycerol-3-phosphate dehydrogenase, and glycerol-3-phosphatase;        and/or    -   (x) 2-phenylethanol biosynthetic enzymes including        aromatic-L-amino acid decarboxylase, 2-phenylethylamine oxidase        and aryl alcohol dehydrogenase.    -   (xi) pHBA biosynthetic enzymes including 4-hydroxycinnamoyl-CoA        hydratas/lyase (HCHL) and chorismate pyruvate lyase (CPL).

Any of a number of products may be produced according to the presentinvention. Examples of compounds that may be produced via metabolicengineering of a subject plant include: vanillin(4-hydroxy-3-methoxybenzaldehyde); sorbitol; PHAs; indigo; fructan;lactic acid (2-hydroxypropanoic Acid); adipic acid; 1,3 propanediol,2-phenylethanol and pHBA. These compounds, however, are only examplary,and the present invention is predicated on the use of C4 plants asbioreactors for any compound that can be synthesised in the plant.Accordingly, the present invention is not limited to any one product ormethod for producing the product.

In a particularly preferred embodiment, the present inventioncontemplates a method for accumulating polymers comprising one or morespecies of hydroxyalkanoic acid monomer in a C4 grass, said methodcomprising expressing one or more genetic sequences which encode enzymesrequired for the production of the polymers or a homolog or precursorthereof in a cell of a C4 grass such that PHA polymers accumulate in thecytosol, storage vacuole or plastid or non-plastid organelle of saidcell.

The present invention further contemplates a method for generating aplant which produces PHAs, said method comprising introducing into cellsof said plant a genetic sequence comprising:—

-   -   (i) a nucleotide sequence encoding a phaA or homolog thereof;    -   (ii) a nucleotide sequence encoding phaB or homolog thereof;    -   (iii) a nucleotide sequence encoding phaC or homolog thereof;    -   (iv) a nucleotide sequence encoding phaC1 or homolog thereof;    -   (v) a nucleotide sequence encoding phaG or homolog thereof;    -   (vi) a nucleotide sequence encoding phaJ or homolog thereof    -   (vii) SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12        or a nucleotide sequence having at least 60% identity thereto        after optimal alignment, or capable of hybridizing to SEQ ID        NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12 or a        complementary form thereof under low stringency conditions;    -   (viii) SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID        NO:15 or a nucleotide sequence having at least 60% identity        thereto after optimal alignment, or capable of hybridizing to        SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or a        complementary form thereof under low stringency conditions;    -   (ix) SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18        or a nucleotide sequence having at least 60% identity thereto        after optimal alignment, or capable of hybridizing to SEQ ID        NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a        complementary form thereof under low stringency conditions;    -   (x) SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24        or SEQ ID NO:25 or SEQ ID NO:27 or a nucleotide sequence having        at least 60% identity thereto after optimal alignment, or        capable of hybridizing to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID        NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:27 or a        complementary form thereof under low stringency conditions;    -   (xi) SEQ ID NO:28 or SEQ ID NO:30 or a nucleotide sequence        having at least 60% identity thereto after optimal alignment, or        capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a        complementary form thereof under low stringency conditions;    -   (xii) SEQ ID NO:31 or SEQ ID NO:33 or a nucleotide sequence        having at least 60% similarity thereto or capable of hybridizing        to SEQ ID NO:31 or SEQ ID NO:33 or a complementary form thereof        under low stringency conditions;        and then regenerating a plant from said cells.

A convenient C4 grass for use in the present invention is sugarcane.Sugarcane has certain advantages, which make it a useful crop for use asa bioreactor including, inter alia, its efficient carbon fixation, highbiomass accumulation, rapid growth and natural ability to accumulatelarge quantities of sucrose. Moreover, it achieves this very efficientlyby collecting solar radiation and converting it into a carbon sink (i.e.sucrose). Sugarcane is also a hardy crop, is relatively easy to grow andprovides a large biomass capability. A micropropagation system isalready available and an industry infrastructure in existence.

In another embodiment, the present invention provides a method forgenerating a plant which produces vanillin or a precursor thereof, saidmethod comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a 3-dehydroshikimate        dehydratase;    -   (ii) a nucleotide sequence encoding        catechol-o-methyltransferase;    -   (iii) a nucleotide sequence encoding aryl aldehyde        dehydrogenase;    -   (iv) a nucleotide sequence encoding feruloyl-CoA synthetase;    -   (v) a nucleotide sequence encoding enoyl-CoA hydratase;    -   (vi) a nucleotide sequence encoding enoyl-CoA aldolase; and/or    -   (vii) a nucleotide sequence encoding a homolog of any one of (i)        through (vi)        and then regenerating a plant from said cells.

Another aspect of the present invention contemplates a method forproducing sorbitol in a C4 grass, said method comprising expressing oneor more genetic sequences encoding a glucose-fructose oxidoreductase, incells of a C4 grass such that sorbitol accumulates anywhere in the cellor extracellular matrix of the plant.

In another embodiment, the present invention is directed to a method forgenerating a plant which produces indigo or a precursor thereof, saidmethod comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding genetic sequences encoding        tryptophanase;    -   (ii) a nucleotide sequence encoding L-tryptophan indole lyase;    -   (iii) a nucleotide sequence encoding napthalene dioxygenase;    -   (iv) a nucleotide sequence comprising the R. eutropha bec gene;    -   (v) the nucleotide sequence set forth in Genbank accession        number D14279, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank D14279 under low stringency conditions.    -   (vi) the nucleotide sequence set forth in Genbank accession        number M83949, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank M83949 under low stringency conditions.    -   (vii) the nucleotide sequence set forth in Genbank accession        number AF306552, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank AF306552 under low stringency conditions.        and then regenerating a plant from said cells.

In another embodiment, the present invention relates to a method forgenerating a C4 grass plant which produces a fructan or a precursorthereof, said method comprising introducing into cells of said plant agenetic sequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a fructosyltransferase    -   (ii) a nucleotide sequence encoding a levansucrase;    -   (iii) the nucleotide sequence set forth in Genbank accession        number AY150365, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank AY150365 under low stringency conditions.        and then regenerating a plant from said cells.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces lactic acid or a precursor thereof,said method comprising introducing into cells of said plant a geneticsequence encoding lactate dehydrogenase and then regenerating a plantfrom said cells.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces adipic acid or a precursor thereof,said method comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a 3-dehydroshikimate        dehydratase and/or;    -   (ii) a nucleotide sequence encoding protochatechuate        decarboxylase;    -   (iii) a nucleotide sequence encoding catechol 1,2-dioxygenase;    -   (iv) a nucleotide sequence encoding 3-ketoacyl-ACP synthase;        and/or    -   (v) a nucleotide sequence encoding a homolog of any one of (i)        though (iv)        and then regenerating a plant from said cells.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces 1,3-propanediol or a precursorthereof, said method comprising introducing into cells of said plant agenetic sequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a glycerol dehydratase        and/or;    -   (ii) a nucleotide sequence comprising the dhaB gene from        Klebsiella pneumoniae, or a homolg thereof;    -   (iii) a nucleotide sequence encoding 1,3-propanediol        oxidoreductase;    -   (iv) a nucleotide sequence comprising the dhaT gene from        Klebsiella pneumoniae or homolg thereof;    -   (v) a nucleotide sequence encoding glycerol-3-phosphate        dehydrogenase;    -   (vi) a nucleotide sequence encoding glycerol-3-phosphatase;        and/or    -   (vi) a nucleotide sequence encoding a homolog of any one of (i)        though (vi)        and then regenerating a plant from said cells.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces 2-phenylethanol or a precursorthereof, said method comprising introducing into cells of said plant agenetic sequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a aromatic-L-amino acid        decarboxylase;    -   (ii) a nucleotide sequence encoding 2-phenylethylamine oxidase;    -   (iii) a nucleotide sequence encoding aryl-alcohol dehydrogenase;        and/or    -   (iv) a nucleotide sequence encoding a homolog of any one of (i)        though (iii)        and then regenerating a plant from said cells.

In another aspect, the present invention contemplates a method forgenerating a plant which produces pHBA or a precursor thereof, saidmethod comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding hydroxycinnamoyl-CoA        hydratase/lyase;    -   (ii) a nucleotide sequence encoding chorismate pyruvate lyase;    -   (iii) a nucleotide sequence comprising the ubiC gene from E.        coli, or a homolg thereof; and/or    -   (iv) a nucleotide sequence comprising the HCHL gene from        Pseudomonas fluorescens or homolg thereof;        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

A further aspect of the present invention provides a transfected ortransformed cell, tissue, or organ from a C4 grass, which comprises anucleotide sequence encoding one or more enzymes required for theproduction of a useful product as well as severed or cut parts of agenetically modified plant including stem, flower, seed or otherreproductive parts.

Accordingly, another aspect of the present invention provides agenetically modified C4 grass having cells carrying one or more geneticsequences such that one of the following products in the cytosol,storage vacuole, non-plastid organelle or extracellular matrix of saidcells:

-   -   (i) polyhydroxy-alkanoate polymers    -   (ii) vanillin    -   (iii) sorbitol    -   (iv) indigo    -   (v) fructans    -   (vi) lactic acid    -   (vii) adipic acid    -   (viii) 1,3-propanediol    -   (ix) 2-phenylethanol    -   (x) pHBA

The present invention extends to parts of plants tissue includingleaves, stems, vascular bundles, bark, reproductive material, roots andany extracted liquid (“juice”) from said plant.

In order to direct product accumulation to a desired sub-cellularlocation, particular specific “target sequences” may be incorporatedinto the genetic constructs described above.

A target sequence includes a signal sequence such as a signal sequenceto direct the protein to a plastid, vacuole, mitochondrion, peroxisomeor ontologically related organelles, or other appropriate organ ortissue.

The plants of the present invention may also be further “tagged” with areporter that identifies the plant as a plant bioreactor. Any number ofphysiological or genetic tags would be suitable, and readily identifiedby one of skill in the art.

Accordingly, the present invention contemplates a plant suitable for useas a bioreactor that has been tagged with a genetic sequence whichencodes or comprises a genotypic or phenotypic feature that allowsdifferentiation of the plant bioreactor from a wild-type plant, or whichidentifies the plant as a propietary plant.

The plant-based bioreactor system of the present invention is useful inenabling the production of molecules such as PHAs, pHBA, vanillin,sorbitol, indigo, fructans, lactic acid, adipic acid, 1,3-propanediol,2-phenylethanol, inter alia, by a number of different parties such asdifferent commercial entities. The present invention extends, therefore,to a data processing system to monitor the use of the plants and/or theproduction of target molecules.

Accordingly, another aspect of the present invention contemplates amethod for generating a target molecule in a sucrose-accumulating plant,said method comprising:—

-   -   (i) providing a plant or cells of a plant to a party; and    -   (ii) permitting the party to generate and harvest molecules from        said plant or cells of said plant receiving and processing data        from said party.

The data received from the party includes, for example, numbers ofplants grown and/or harvested, the types of genetic constructsintroduced into the cells and/or income received from sale of theproducts.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation showing the four differentbiosynthetic strategies employed by bacteria that use PHAs as an energysource.

FIG. 2 is a diagrammatic representation of the biosynthetic pathway ofPHB in the bacterium R. eutropha. This pathway comprises three stepscatalyzed, respectively, by three enzymes: first, two molecules ofacetyl-CoA are condensed to acetoacetyl-CoA by 3-ketothiolase (encodedby phaA); secondly, acetoacetyl-CoA is reduced to D-3-hydroxybutyryl-CoAby acetoacetyl-CoA reductase (phaB); and thirdly, D-3-hydroxybutyryl-CoAis polymerized to PHB by PHB synthase (phaC).

FIG. 3 is a diagrammatic representation of the flow of carbon insub-cellular compartments within a sugarcane cell.

FIG. 4 shows a graphical representation of the detection of PHB inchloroplasts of transgenic sugarcane. A-C: Detection of PHB by HPLC. A:WT sugarcane (s/c) −ve control; B: sample in A spiked with PHB; C:plastid-targeted, PHB +ve s/c line. Arrows: elution point of crotonicacid (PHB breakdown product). Insert in C shows that the peak at 30 minin C has the same spectrum as crotonic acid. D-F: Detection of PHBgranules by transmission electron microscopy. D: Chloroplast (c/p) ofmesophyll cell of PHB +ve Arabidopsis control; E: c/p of mesophyll cellof PHB +ve s/c line; F: c/p of bundle-sheath cell of same line in E.Scale bars=200 nm.

FIG. 5 illustrates the agronomic performance of PHB-producing sugarcanelines. Four transgenic sugarcane lines expressing the PHB biosynthesisgenes of R. eutropha (filled bars) were grown for 3 months in arandomised glasshouse plot, together with GFP expressing (open bars) andtissue-culture-regenerated WT (hatched bars) plants as controls. PHBcontent in lamina from the tips of mature leaves was quantified by HPLCanalysis. Data are the mean±SE (n=3). DW=dry-weight.

FIG. 6 shows the affect of PHB production on sugarcane sugaraccumulation. Mature-(A-D) and intermediate-aged (E-H) stem internodesfrom the PHB producing (solid bars), GFP expressing (open bars) and WT(hatched bars) sugarcane plants in FIG. 5 were assayed for sucrose,glucose and fructose concentrations. Data are the mean±SE (n=3).DW=dry-weight.

FIG. 7 is a graphical representation showing the distribution of PHBthroughout a PHB-producing sugarcane line. The distribution of PHBthroughout transgenic sugarcane line PHB3 in FIG. 5 was determined byHPLC analysis. Samples were taken from: lamina of the tip, midpoint andbase of young, intermediate and mature leaves; rind+pith of young,intermediate and mature stem internodes; and roots. Data are the mean %of leaf DW as PHB±SE (n=3). ND=not detected.

FIG. 8 is a graphical representation showing the indigo biosyntheticpathway.

FIG. 9 is a graphical representation showing adipic acid biosynthesisfrom glucose via a cis, cis-muconic acid intermediate.d=3-dehydroshikimate dehydratase, e=protochatechuate decarboxylase,f=catechol-1,2-dioxygenase.

FIG. 10 is a graphical representation showing adipic acid biosynthesisby petroselinic acid ozonolysis.

FIG. 11 is a graphical representation showing 2-phenylethanolbiosynthesis.

FIG. 12 is a graphical representation showing the basic steps of C4carbon assimilation.

FIG. 13 is a graphical representation depicting the detection of pHBAand vanillate by HPLC in acid hydrolysed leaf samples taken fromsugarcane leaves expressing the HCHL transgene. A: Indicated peaks showthe presence of pHBA and vanillate in the leaf extract. Insets show thecharacteristic spectrum profiles for the respective compounds. B: Peaksproduced by pHBA and vanillate synthetic standards.

FIG. 14 is a graphical representation of p-hydroxybenzoic acid (pHBA)synthesis in planta can be accomplished by the introduction of E. colichorismate pyruvate-lyase (CPL) or P. fluorescens 4-hydroxycinnamoyl-CoAhydratase/lyase (HCHL). CPL converts plastidal chorismate into pHBAwhilst HCHL converts cytosolic 4-coumaroyl-CoA into4-hydroxybenzaldehyde via a â-hydroxy thioester intermediate which issubsequently oxidized to pHBA by endogenous NAD+-linked dehydrogenases.In both instances, the majority of the resultant free acid isglucosylated and transported into vacuoles. Abbreviations: E4P,erythrose-4-phosphate; PEP, phosphoenolpyruvate; UDP-GT,UDP-glucosyltransferase.

FIG. 15 is a graphical representation showing the distribution patternof p-hydroxybenozic acid (pHBA) in UH1 at 20 weeks. (a) Leaf andinternode pHBA levels are compared. There is generally more pHBA in theleaf than the stalk and the content in older tissue is generally higherthan younger tissue. (b) pHBA levels at specific locations in the tissueare compared. The largest quantities of pHBA are found in the leaflamina and the rind tissue of the stem. (LL=leaf lamina; LM=leaf midrib;R=rind; P=pith; VB=vascular bundles of stem tissue).

FIG. 16 is a photographic representation showing a comparison of thegrowth phenotype between the highest pHBA producer, UH98 and the controlline TC1 reveals no obvious differences. (a) Plants of approximatelyequivalent age were compared. TC1, (left), UH98, (right). Inset: Aclose-up view of the under surface of a leaf is shown for TC1 (b) andUH98 (c).

A summary of sequence identifiers used throughout the subjectspecification is provided below in Table 1:

TABLE 1 SUMMARY OF SEQUENCE IDENTIFIERS SEQUENCE ID NO: DESCRIPTION 1Nucleotide sequence (phaA) encoding PhaA without signal sequence (hence,the PhaA remains in the cytosol) 2 Amino acid sequence of PhaA withoutsignal sequence 3 Nucleotide sequence (phaA) encoding PhaA withoutsignal sequence (hence, the PhaA remains in the cytosol) modified at 5′and 3′ ends for insertion into a vector 4 Nucleotide sequence (phaB)encoding PhaB without signal sequence (hence, the PhaB remains in thecytosol) 5 Amino acid sequence of PhaB without signal sequence 6Nucleotide sequence (phaB) encoding PhaB without signal sequence (hence,the PhaB remains in the cytosol) modified at 5′ and 3′ ends forinsertion into a vector 7 Nucleotide sequence (phaC) encoding PhaCwithout signal sequence (hence, the PhaC remains in the cytosol) 8 Aminoacid sequence of PhaC without signal sequence 9 Nucleotide sequence(phaC) encoding PhaC without signal sequence (hence, the PhaC remains inthe cytosol) modified at 5′ and 3′ ends for insertion into a vector 10Nucleotide sequence (phaA) encoding PhaA targeted to plastid 11 Aminoacid sequence of PhaA with signal sequence to target to the plastid 12Nucleotide sequence (phaA) encoding PhaA targeted to plastid modified at5′ and 3′ ends for insertion into a vector 13 Nucleotide sequence (phaB)encoding PhaB targeted to plastid 14 Amino acid sequence of PhaB withsignal sequence to target to the plastid 15 Nucleotide sequence (phaB)encoding PhaB targeted to plastid modified at 5′ and 3′ ends forinsertion into a vector 16 Nucleotide sequence (phaC) encoding PhaCtargeted to plastid 17 Amino acid of PhaC with signal sequence to targetto the plastid 18 Nucleotide sequence (phaC) encoding PhaC targeted tomodified at 5′ and 3′ ends for insertion into a vector 19 Nucleotidesequence (phaC1) encoding PhaC1 without signal sequence (hence, thePhaC1 remains in the cytosol) 20 Amino acid sequence of PhaC1 withoutsignal sequence 21 Nucleotide sequence (phaC1) encoding PhaC1 withoutsignal sequence modified at 5′ and 3′ ends for insertion into a vector22 Nucleotide sequence (phaC1) encoding PhaC1 targeted to the peroxisome23 Amino acid sequence of PhaC1 with signal sequence to target to theperoxisome 24 Nucleotide sequence (phaC1) encoding PhaC1 targeted to theperoxisome modified to 5′ and 3′ ends for insertion into a vector 25Nucleotide sequence of (phaC1) encoding PhaC1 targeted to the plastid 26Amino acid sequence of PhaC1 with signal sequence to target to theplastid 27 Nucleotide sequence (phaC1)) encoding PhaC1 targeted to theplastid modified at 5′ and 3′ ends for insertion into a vector 28Nucleotide sequence (phaG) encoding PhaG targeted to the plastid 29Amino acid sequence of PhaG with signal sequence to target to theplastid 30 Nucleotide sequence (phaG) encoding PhaG targeted to theplastid modified to 5′ and 3′ ends for insertion into a vector 31Nucleotide sequence (phaJ) encoding PhaJ targeted to the peroxisome 32Amino acid sequence of PhaJ with signal sequence to target to theperoxisome 33 Nucleotide sequence (phaJ) encoding PhaJ targeted to theperoxisome modified to 5′ and 3′ ends for insertion into a vector 34TphaF 35 PhaF 36 PhbF 37 PhcF 38 PhaR 39 PhbR 40 PhcR 41 PhaC1Cf 42PhaC1Cr 43 PhaC1PF 44 PhaJF 45 PhaJR 46 PhaGF 47 PhaGR 48 SSP-F 49 SSP-R50 primer 3 51 primer 4 52 primer 5 53 primer 6

A list of abbreviations used herein is provided in table 2.

TABLE 2 Abbreviations ABBREVIATION Description 1,3-PD 1,3-Propanediol2-PE 2-Phenylethanol CAM Crassulacean Acid Metabolism DW Dry weight GFORGlucose-Fructose Oxidoreductase GFP Green Fluorescent Protein PEPPhosphoenolpyruvate PEPcase Phosphoenolpyruvate carboxylase PETPolyethylene terephthalate sPET Isosorbide PET PHA PolyhydroxyalkanoatePHB Polyhydroxybutyrate pHBA poly hydroxybenzaldehyde PHVPolyhydroxyvalerate/Poly-3-hydroxypentanoate Rubisco Ribulosebisphosphate carboxylase/oxygenase WT Wild type

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a plant-derived bioreactor system, whichis capable of withstanding the extra metabolic load placed on individualplants without resulting in deleterious growth effects. Hence, until theadvent of the present invention, plants have been engineered to makesmall quantities of a desired product, their capacity to do so beinglimited, and their ability to go on doing so prevented, by thesedeleterious growth effects. In accordance with the present invention,certain plants were selected on the basis of the presence of highlyefficient photosynthetic mechanisms for the assimilation of carbon,particular metabolic reserves, and/or useful metabolic and/orbiosynthetic pathways. Grasses, and particularly C4 grasses, wereidentified as having particularly efficient mechanisms for theassimilation of carbon, a high growth rate and high accumulation ofbiomass and hence are useful as bioreactors for the production of a widerange of products. Furthermore, the C4 grass, sugarcane, is particularlyuseful, as this plant stores sugars in dimeric and/or polymeric formsthat may be utilized when needed, for example, to supply energy duringtimes of significant environmental stress. This store of carbohydratewould provide a ready supply of substrate for many metabolic pathways,and utilization of this store would not stress the producing plant.Therefore, the present invention is predicated, in part, on theidentification of a subset of plants, namely the C4 grasses, andparticularly sugarcane, as useful bioreactors on the basis of their highcarbon assimilation rate, rapid growth, high biomass production andlarge store of carbohydrate.

Accordingly, one aspect of the present invention provides a method forgenerating a plant-based bioreactor system, said method comprisingselecting a plant having a high efficiency carbon assimilationmechanism, rapid growth rate and/or high biomass production and/orreserves of metabolites or having a capacity to generate such reservesand/or which possess metabolic and/or biosynthetic pathways useful inthe manufacture of a product of interest or a precursor form thereof;genetically modifying cells of the plant to enable access to saidmetabolites and/or metabolic or biosynthetic pathways; and thenregenerating a genetically modified plant from said cells.

The terms “grass”, “grasses” and the like are to be understood asreference to any member of the Gramineae plant family whether currentlyknown or not. This family currently encompasses approximately 660 plantgenera including 10,000 plant species. It will be readily apparent tothe skilled artisan when examining a given plant, either known or novel,whether the plant is a grass.

Preferably, the grass is a C4 grass.

To minimise losses of carbon and nitrogen resulting fromphotorespiration, some plants such as corn and sugarcane that grow inhot climates have a different system for fixing CO₂, called C4photosynthesis, than plants that grow in more temperate climates (whichhave C3 photosynthesis). The leaf anatomy of plants such as corn andsugarcane is different from that of temperate plants. The vascularbundles of these leaves are surrounded by a wreath of thick-walledparenchyma cells called bundle sheath cells, where most of thecarbon-fixation takes place.

During C4 photosynthesis, CO₂ in the mesophyll cells is condensed with a3C compound called phosphoenolpyruvic acid (PEP), by the action of theenzyme PEP carboxylase. This produces the 4C compound oxaloacetic acidwhich is then converted to malic or aspartic acid. The malic or asparticacid is then moved through plasmodesmata (at the expense of ATP) intothe bundle sheath cells.

In the bundle sheath cells, the 4C compounds are decarboxylated torelease CO₂ and PEP. The CO₂ collected in the many mesophyll cells isconcentrated into a few bundle sheath cells. Therefore, the plants canmaintain a higher concentration of CO₂ in the bundle sheath cells (wherethe Calvin-Benson cycle of photosythesis occurs) than it can elsewherein the leaf. This higher concentration of CO₂ minimisesphotorespiration.

The C4 pathway is more expensive energetically than C3 photosynthesis,but this is offset by the resulting decrease in photorespiration (whereunder certain conditions, plants may lose 30% of fixed carbon). For thisreason, C4 plants are well adapted to environments that promote highlevels of photorespiration (viz. subtropical and tropical climes).

The fundamentals of C4 photosynthesis are shown schematically in Figure*. The photosynthesis processes of C4 plants are divided betweenmesophyll and bundle sheath cells. Two steps of C4 photosynthesis whichoccur in the mesophyll cells are the light-dependent reactions and apreliminary fixation of CO₂ into malate or aspartate (a 4C compound).This C4 compound is transported to the bundle sheath cells, and isdecarboxylated to form CO₂ and PEP. The released CO₂ is re-fixed byRubisco and the Calvin-Benson cycle. The PEP is then recycled back tothe mesophyll cells, and the photosynthates are distributed throughoutthe plant.

One defining aspect of a C4 plant is “Kranz anatomy” (German for“wreath”). This term refers to the characteristic one, or twoconcentric, layer(s) (wreath[s]) of bundle sheath cells, around thevascular bundles of the leaves. The mesophyll cells surrounding thebundle sheath cells fix CO₂ via PEP carboxylase to form 4C organicacids. These are transported to the bundle sheath cells and aredecarboxylated to regenerate CO₂, which is then refixed by the typicalC3 photosynthetic pathway found in non-C4 plants. The system thus actsas a CO₂ pump, increasing the CO₂ concentration in the bundle sheath toa level where photorespiration is minimised.

Accordingly, for the purposes of the present invention C4 plants are tobe understood as plants which exhibit at least one of the followingcharacteristics in at least some part of the plant:

-   -   (i) Kranz anatomy;    -   (ii) fixation of CO₂ into a 4 carbon compound; and/or    -   (iii) decarboxylation of a 4 carbon compound.

For the purposes of the present invention, a given plant need notexhibit all of the above characteristics to be considered a C4 plant.For example, Borszczowia aralocaspica (Chenopodiaceae) has thephotosynthetic features of C4 plants, yet lacks Kranz anatomy. Thisspecies accomplishes C4 photosynthesis through spatial compartmentationof photosynthetic enzymes, and by separation of two types ofchloroplasts and other organelles in distinct positions within thechlorenchyma cell cytoplasm. Accordingly, insofar as the presentinvention relates to C4 plants, this is to be understood as those whichexhibit 1, 2 or all 3 of the above characteristics.

It is also to be understood that the subject plant need not exhibit oneor all of these criteria at all times. Some plant species, such as theamphibious leafless sedge, Eleocharis vivipara, can exhibit C3 and C4characteristics such as those shown above depending on whether it isgrown in a terrestrial or aquatic environment. In addition, the Cassavaplant has photosynthetic mechanisms which are typical of a C3 plant, yetsome studies have shown that both C3 and C4 enzymatic systems functionin Cassava. The dominant photosynthetic pathway varies between C3 and C4depending on temperature: at lower temperatures, photosynthesis followsa C3 path, and at higher temperatures, a C4 path.

Therefore, for the purposes of the present invention, C4 plants are tobe understood as those plants capable of exhibiting one or more of theabove characteristics under any given environmental condition. Thepresent is not limited by the method of photosynthesis used at a giventime by a given plant, the plant need only be capable of expressing atleast one of the above characteristics associated with C4photosynthesis.

In addition, plants utilising the Crassulacean Acid Metabolism (CAM)pathway are also to be considered within the definition of a C4 plantfor the purposes of the present invention.

The term “Crassulacean” refers to the Stonecrop family (Crassulaceae)and related succulents in which this process is common. To date, plantsin more than 18 different families including Cactaceae (Cactus family)and Bromeliaceae (Pineapple family) have been shown to carry out CAMmetabolism. The term “Acid” is derived from the observation that theseplants accumulate large amounts of C4 organic acids in the dark.

Plants with CAM metabolism are typically adapted to dry, hot, high-lightenvironments. CAM is largely a mechanism to conserve water. Plants indry environments utilise CAM as they cannot afford to lose water byopening their stomata during the day. CAM plants circumvent water lossduring the day by opening up the stomates at night to obtain carbondioxide.

Carbon dioxide is accumulated in CAM plants using PEP carboxylase, andthe fixed carbon is stored as 4-carbon compounds such as malate, as inC4 plants.

The CO₂ obtained during the night is stored as a C4 acid until ATP andNADPH are available the following day as a result of the light reactionsof photosynthesis. The C4 acid is then decarboxylated and the CO₂ fixedby the Calvin-Benson cycle. Thus, in CAM plants there is a temporalseparation of initial carbon fixation and the Calvin-Benson cycle,whereas in other C4 plants there is a spatial separation.

In summary the sequence of events in CAM plants is:

Night→stomates→open nocturnal transpiration (lower than diurnal) andcarbon fixation by PEPcase→OAA produced→reduced with NADPH tomalate→shuttled into vacuole as malic acid→malic acid content of vacuoleincreases→starch depleted to provide PEP for carboxylation→day→stomatesclose→transpiration decreased→malic acid content decreases→resultingmalate decarboxylated to provide carbon dioxide for Calvin cycle→starchcontent increases.

Accordingly, as CAM plants exhibit fixation of CO₂ into a 4C compound,and decarboxylation of a 4C compound, they are to be understood aswithin the definition of C4 plants for the purposes of the presentinvention.

The present invention is particularly directed to C4 grasses; however,other non-grass C4 plants such as woody or herbaceous plants whichutilise the C4 pathway are also contemplated by, and are within thescope of, the present invention.

In a preferred embodiment of the present invention the subject plant isa grass, more preferrably a C4 grass. In a particularly preferredembodiment the C4 grass is a member of the Saccharum genus, andparticularly the Saccharum hybrid, sugarcane.

Commercially grown sugarcane varieties are mainly interspecific hybridsand are vegetatively propagated. There are about six different speciescontributing to the gene pool: Saccharum officinarum, Saccharumrobustum, Saccharum barberi, Saccharum spontaneum, Saccharum sinense andErianthus sp. Hence, the scope of present invention is not to be limitedto any one variety but should be regarded as extending to andencompassing other species of Saccharum.

The preferred compounds to be produced by the plant bioreactor include:vanillin, sorbitol, PHAs, indigo, fructan, lactic acid, adipic acid,1,3-propanediol and 2-phenylethanol. However, the present inventionextends to the use of C4 grasses as bioreactors to generate a compoundssuch as therapeutics, nutrapharmaceuticals, diagnostic agents includingsingle chain antibodies, industrial enzymes and the like. However, thepresent invention is in no way limited by the exemplified compounds andmethods.

The present invention contemplates, therefore, a method for producing aproduct of interest including a product or intermediate of abiosynthetic or metabolic pathway in a C4 grass, said method comprisingexpressing one or more genetic sequences which encode one or moreenzymes or proteins required for the production of the product orintermediate or a homolog or precursor thereof or which induces genesilencing of genetic material which encodes an enzyme or protein in abiosynthetic or metabolic pathway in cell of a C4 grass plant such thatthe product or intermediate accumulates in the cytosol, storage vacuole,plastid or non-plastid organelles of the cell, or accumulates in thejuice or vascular fluid of the plant.

For the purposes of the present invention, the application of a plant asa bioreactor, is to be also understood as the alteration of existingplant metabolism or the introduction of new plant metabolism to generatea non-endogenous plant product or an endogenous plant product atnon-native levels.

In the case of the metabolic engineering of native plant biochemicalpathways, this may be achieved via a number of means. Alterations to themetabolic activity of an organism can be made at the gene, geneexpression and protein levels.

Metabolic engineering may be affected at the protein level in anorganism by the administration of particular enzyme activators orinhibitors. For example, the activity of particular biosynthetic enzymesmay be regulated by the administration of particular enzyme inhibitorsto the plant. These inhibitors may directly effect the accumulation of aproduct by reducing the activity of a particular biosynthetic enzyme. Inaddition indirect effects such as the redirection of metabolic flux intoother pathways may be a product of a particular enzyme inhibition. Forexample, the blocking of a particular enzyme may lead to the buildup ofan intermediate which may then be directed into a different metabolicpathway, leading to the increased accumulation of the product of thesecond pathway.

The present invention therefore contemplates the production of a productin a C4 grass wherein product accumulation is at least in partpredicated on the direct activation or inhibition of an enzyme in abiosynthetic pathway by the administration to the plant of an enzymeinhibitor or activator. An enzyme “inhibitor” or “activator” alsoincludes genetic inhibitors or activators; ie. nucleic acid molecules orRNAi-type molecules which induce gene silencing or a structural gene ora regulatory gene which positively or negatively regulates structuralgene expression.

In a preferred embodiment, increased accumulation of a product orintermediate from a biosynthetic or metabolic pathway is a result ofinhibition of one or more biosynthetic enzymes in the pathway orre-direction of metabolite flow down another pathway.

Particular biosynthetic or metabolic pathways may be induced in plants,parts of plants and/or plant cells in culture by the addition ofelicitors, or by changes in environmental conditions. Gene expression inplants and other organisms is mediated by a number of physical, chemicaland biotic factors. Physical factors such as light intensity andphotoperiod have been implicated in the expression of many plant genesincluding genes involved in morphogenesis and plant secondarymetabolism. For example, the anthocyanin biosynthetic genes, PAL and CHSare induced by increased light intensity and increased photoperiod. In asimilar way, temperature and osmotic stresses have also been shown toalter gene expression in a broad range of biological systems. Forexample, the KIN1, COR15a, and LTI78 genes in Arabidopsis thaliana aresensitive to induction by low temperatures (Knight et al., Plant Cell11(5): 875-886, 1999), and a range of heat shock proteins and glycolyticenzymes are induced in the bacterium Lactobacillus rhamnosus in responseto heat and osmotic stress (Prasad et al., Appl. Environ. Microbiol.69(2): 917-925, 2003). Many of these gene expression changes are aresult of stress to the cells. In addition, other physical factors suchas wounding and drought have also been associated with altered geneexpression in plants. For example in the fig tree Ficus carica, droughtstress induced genes encoding a peroxidase, a chitinase and a trypsininhibitor (Kim et al., Plant Cell Physiol. 44(4): 412-414).

Chemical inducers of gene expression have been identified for manybiological systems and specific gene promoters. Examples of theseinclude the induction of chalcone synthase, a phenylpropanoid pathwayenzyme, by chemical elictors such as jasmonate. Also, bacteriallysynthesized lipochitooligosaccharides (Nod factors) induce the earlynodulin (ENOD) genes in leguminous plants (Fang and Hirsch, PlantPhysiol. 115:53-68, 1998). Several compounds have also been demonstratedto induce gene expression associated with plant defence, such as silicondioxide, phosphate salts, and polyunsaturated fatty acids (Sticher etal., Annu. Rev. Phytopathol. 35: 235-270, 1997).

A number of biological agents have also been demonstrated to alter geneexpression in other organisms. For example, many phytopathogenic fungiand bacteria induce the expression of a number of defence-related genes,such as the PR proteins, β-glucanases, terpenoid biosynthetic enzymesand genes in the ‘salicylic acid’ defence pathway. Non-pathogenic,microbial colonists of plant induce yet another different set of genesin the plant host (Han et al., Phytopathology 90: 327-332, 2000).

Alterations or changes to cultural practices, culture conditions andgrowth conditions, including photoperiod and/or temperature, areconsidered to be conditions which are not standard conditions for thegrowth of the plant or cell. For example, growth of a plant or plantcell culture under a 24 hour light, would be considered an alteredphotoperiod for the purposes of the present invention. Second, growth ofa plant at a temperature substantially above or below the optimum growthtemperature of said plant, plant cell culture or bacterial culture wouldbe considered an altered temperature for the purposes of the presentinvention. The preceeding examples in no way limit the invention and itwill be clear to those of skill in the art what constitutes altered,changed or abnormal conditions pertaining to a given cell, cell culture,plant or organism.

Methods of altering gene expression in sugarcane contemplated by thepresent invention are to be understood as physical processes orconditions, chemical compounds and biological, including genetic agents.Non-limiting examples of physical agents include alterations to cultureand/or growth conditions of the cell or organism, light intensity and/orphotoperiod, temperature, growing season, and or physical wounding.Examples of chemical agents that may alter gene expression in plantsinclude phytohormones such as auxins, cytokinins and gibberellins;signalling molecules such as flavanoids, saccharides, sterols andpeptides; herbicides and antibiotics. Examples of biological agentscontemplated by the present invention include microorganisms, such asbacteria and fungi; viruses; transposons and plasmids as well asRNAi-inducing genetic molecules including a hairpin loop or other meansto induce gene silencing (eg. post-transcriptional gene silencing). Thepreceeding examples are only illustrative in nature and in no way limitthe invention to the said agents.

Accordingly, the present invention contemplates the use of sugarcane asa bioreactor. Therefore, alteration to the gene expression profile ofsugarcane to effect the production of an endogenous metabolite at anincreased level, or to produce any heterologous metabolite is within thescope of the present invention. Accordingly, induction or supression ofany biosythetic or metabolic genes in sugarcane, such as thoseexemplified herein, is to be considered within the scope of the presentinvention. Reference to a biosynthetic or metabolic gene also includes aregulatory gene.

The application of a plant as a bioreactor may be affected by theintroduction of a new biosynthetic or metabolic pathway into the plant,or the redirection of metabolic flux down a particular pathway in aplant. For the purposes of the present invention, “introduction of a newbiosynthetic or metabolic pathway” encompasses where the introducedpathway is a single protein or enzyme, which may in itself be theend-product. For example, the introduction of a nucleic acid molecule,whether or not it encodes a protein of interest, would fall within thescope of the subject invention. In this regard, a “protein” includes aprotein, polypeptide or peptide as well as a glycoprotein,phosphoprotein or phospholipoprotein. Alternatively, the invention alsocontemplates the introduction of one or more enzymes or proteins,wherein the introduced enzyme or protein catalyses one or more reactionsin the synthesis of the product of interest. For example, inplantasynthesis of vanillin could be introduced to sugarcane by theintroduction of the enzymes feruloyl-CoA synthetase and enoyl-CoAhydratase.

Typically, the production of one or more metabolites or heterologousproteins, polypeptides or peptides in a plant is achieved by expressionof a nucleic acid molecule encoding the protein, polypeptide or peptideof interest. Any nucleic acid which encodes a protein, polypeptide orpeptide of interest is contemplated by the present invention. However,preferred nucleic acids include those encoding:

-   -   (i) vanillin biosynthetic enzymes, including 3-dehydroshikimate        dehydratase, catechol-o-methyltransferase, aryl aldehyde        dehdrogenase, feruloyl-CoA synthetase, enoyl-CoA        hydratase/aldolase;    -   (ii) sorbitol biosynthetic enzymes, including glucose/fructose        oxidoreductase;    -   (iii) PHA biosynthetic enzymes, including 3-ketothiolase,        acetoacetyl-CoA reductase, PHA synthase, enoyl hydratase,        3-hydroxyacyl-acyl carrier protein:CoA tranferase;    -   (iv) indigo biosynthetic enzymes, including tryptophanase,        L-tryptophan indole lyase, napthalene dioxygenase, R. eutrophica        bec gene product;    -   (v) fructan biosynthetic enzymes, including        fructosyltransferases and levansucrases;    -   (vi) lactic acid biosynthetic enzymes, including lactate        dehydrogenase;    -   (vii) adipic acid biosynthetic enzymes, including        3-dehydroshikimate dehydratase, protocatechuate decarboxylase        and catechol 1,2-dioxygenase;    -   (viii) petroselinic acid biosynthetic enzymes, including        3-ketoacyl-ACP synthase;    -   (ix) 1,3-propanediol biosynthetic enzymes including glycerol        dehydratase, 1,3-propanediol oxidoreductase,        glycerol-3-phosphate dehydrogenase, and glycerol-3-phosphatase;        and/or    -   (x) 2-phenylethanol biosynthetic enzymes including        aromatic-L-amino acid decarboxylase, 2-phenylethylamine oxidase        and aryl alcohol dehydrogenase.    -   (xi) pHBA biosynthetic enzymes including 4-hydroxycinnamoyl        hydratase/lyase and chorismate pyruvate lyase.

Nucleic acids encoding a particular protein or enzyme may be chemicallysynthesised or isolated from another organism. In a preferred embodimentof the present invention, the gene encoding a protein or enzyme ofinterest is isolated from bacteria, fungi, animals, plants, protists orarchaea. Bacteria provide a convenient source of genes encoding usefulenzymes and proteins, although the present invention should not belimited by the source of the gene encoding the protein or enzyme ofinterest. Particularly useful microorganisms for the isolation of usefulgenes in the context of the present invention include: R. eutropha,Aeromonas spp., Pseudomonas aeruginosa, Rhodococcus ruber, Nocardiacorallina, Zymomonas mobilis, Enterobacter aerogenes, Pseudomonasputida, Bacillus subtilis, Klebsiella pneumoniae, Acinetobactercalcoaceticus the actinomycetes (particularly Streptomyces spp.),Escherichia coli and yeast such as Saccharomyces cereviseae. However, itshould be noted that these microorganisms represent only examples of thepossible source of a gene encoding a protein or enzyme of interest, andthe present invention is in no way limited by the source of the nucleicacid encoding the protein or enzyme of interest. Furthermore, asindicated above, the nucleic acid molecule may not necessarily encode anenzyme or protein but may encode a sense RNA or an antisense RNA, foruse in gene silencing for example.

In order to maximise transcription, and/or transcript stability and/ortranslation and/or post-translational stability of the gene product of aheterologous gene in a plant, particularly a gene from a bacterium, itmay be necessary to alter the sequence of the gene. For example, tomaximise translation of the gene transcript, it may be necessary toalter the coding sequence of the gene to reflect the preferred codonusage of the host plant. Similarly, to maximise translation of the genetranscript it may be necessary to alter the sequence context of thegene's translation initiation site to reflect the preferred sequencecontext recognised by the host's translational machinery. Similarly, itmay be necessary to add 5′ and/or 3′ non-translated regions to thecoding sequence of the gene to maximise transcript stability within thehost cell. Similarly, it may be necessary to alter the coding sequenceof the gene to remove cryptic protease-recognition sites. Therefore, thepresent invention encompasses genes isolated from a bacterial, fungal,animal, protist or archaeal source, which have undergone modification tomaximise transcription, and/or transcript stability, and/or translation,and or post-translational stability in a plant host. Other methods forthe alteration of the sequence of a gene will be readily ascertained bythose of skill in the art and need not be elaborated further here.

In a preferred embodiment of the present invention, a number of productsmay be produced according to any of a number of methods including thoseherein described. Examples of compounds that may be produced viametabolic engineering of a subject plant include: vanillin(4-hydroxy-3-methoxybenzaldehyde); sorbitol; PHAs; indigo; fructan;lactic acid (2-hydroxypropanoic Acid); adipic acid; 1,3 propanediol and2-phenylethanol. These compounds, however, are only examplary, and thepresent invention is predicated on the use of C4 plants as bioreactorsfor any compound that can be synthesised in the plant. Accordingly, thepresent invention is not limited to any one product or method forproducing the product.

In a preferred embodiment of the present invention, the plant selectedis a C4 grass and the product of interest is a mixture of differentchain length polymers of hydroxyalkanoic acid monomers. The presentinvention extends, however, to the use of C4 grasses as bioreactors togenerate a range of compounds such as therapeutics, nutrapharmaceuticalsand diagnostic agents such as single chain antibodies.

Accordingly, the present invention contemplates a method foraccumulating polymers comprising one or more species of hydroxyalkanoicacid monomer in a C4 grass, said method comprising expressing one ormore genetic sequences which encode enzymes required for the productionof the polymers or a homolog or precursor thereof in a cell of a C4grass such that PHA polymers accumulate in the cytosol, storage vacuole,peroxisome and ontologically related organelles, or plastid ornon-plastid organelles of said cell.

Polyhydroxyalkanoates (PHAs) are polyesters of one or more species ofhydroxyalkanoic acid monomers. PHAs, which are bacterial carbon-storagepolymers analogous to starch in plants and glycogen in animals, are adiverse class of compounds, with over 100 different hydroxyalkanoic acidsidechains identified to date. For example, PHB is a polymer of3-hydroxybutyrate, and PHV is a polymer of hydroxyvalerate. PHAs canalso be co-polymers, for example,poly-(3-hydroxybutyrate-co-3-hydroxyvalerate). As defined herein,therefore, “polymers comprising one or more species of hydroxyalkanoicacid monomer” and “PHAs” are synonymous and encompass any suchcarbon-storage polymer.

PHAs are polymers that share many properties with petrochemicallyderived, synthetic polymers. The main advantages of PHAs over syntheticpolymers are that they are readily biodegradable and are made fromrenewable resources such as sugars and fatty acids.

While different bacterial species have developed different mechanismsfor PHA biosynthesis, 3-hydroxyacylCoA is the precursor for them all.FIG. 1 outlines four different strategies utilised by a wide range ofbacteria including, for example, R. eutropha, Aeromonas spp.,Pseudomonas aeruginosa, Rhodococcus ruber and Nocardia corallina, tosynthesize their requirements of polymers of 3-hydroxy acids as anenergy source. Precursors may be generated by sugar glycolysis (strategyI), from metabolic intermediates of the β-oxidative (strategy II) andbiosynthetic fatty acid (strategy III) pathways and from metabolites inother pathways such as the methylmalonyl-CoA pathway (strategy IV).

In the bacterium R. eutropha, for example, strategy I is used and thePHB biosynthetic pathway consists of three steps catalyzed by threeenzymes, respectively: first, two molecules of acetyl CoA are condensedto acetoacetyl-CoA by 3-ketothiolase (encoded by phaA); secondly,aceotacetyl-CoA is reduced to D-3-hydroxybutyryl-CoA by acetoacetyl-CoAreductase (encoded by phaB); and thirdly, D-3-hydroxybutyryl-CoA ispolymerized into PHB by PHB synthase (encoded by phaC). The genes areclustered in a single operon in the order pha-CAB A diagrammaticrepresentation of this particular biosynthetic pathway is provided inFIG. 2.

Aeromonas spp. are examples of organisms that employ strategy II. Theyexpress a (R)-specific enoyl hydratase (PhaJ). This enzyme catalyzes theformation of 3-hydroxyacyl-CoA from enoyl-CoA intermediates inβ-oxidation of fatty acids, thereby generating substrates for thepolymerase. Alternatively, organisms like P. aeruginosa produce PHAsfrom intermediates in the de novo fatty acid biosynthetic pathway(strategy III). A 3-hydroxyacyl-acyl carrier protein:CoA transferaseenzyme designated PhaG converts 3-hydroxyacyl-acyl carrier protein toits CoA analog. The hydroxyacyl-CoA is then incorporated into thenascent polymer. Finally, organisms such as R. ruber and N. corallinaare able to generate precursors for PHA production from themethylmalonly-CoA pathway (strategy IV).

Many other micro-organisms have developed alternative biosyntheticpathways for the manufacture of PHAs, including PHB, to meet theirenergy-requiring needs. Biosynthetic genes from almost 40 differentorganisms have been cloned. Only limited homologies are exhibited atboth the nucleotide and amino acid levels, which is not surprisingconsidering the large number of PHAs naturally produced by bacteria. Thestructural organisation of loci encoding PHA genes is equally diverse.

Any one or more or a combination of these systems may be adapted toprovide suitable genetic sequences for use in accordance with thepresent invention. Consequently, genetic sequences which “encode enzymesrequired for the production of polymers” of hydroxyalkanoic acids asused herein in the context of the present invention may comprise acombination of one or more of any sequence wherein the enzyme or enzymesthereby encoded usually operate in vivo singly or together to effect thebiosynthesis of PHAs, including PHB.

Preferred suitable genetic sequences comprise a combination of one ormore of phaA, phaB, phaC, phaC1, phaG, phaJ. These genes encode enzymeproducts referred to herein as PhaA, PhaB and PhaC, PhaC1, PhaG andPhaJ.

In one preferred embodiment, the C4 grass is engineered to express oneor more of phaA, phaB and phaC such that it does not accumulate the PHAsin the plastid. In another preferred embodiment, the plant is engineeredto express, in addition, one or more of phaC1, phaG and phaJ, such thatit accumulates the PHAs in the plastid.

The nucleotide sequences encoding phaA, phaB and phaC may come from anysuitable source but the genes from R. eutrophia are particularly usefulin the practice of the present invention. The nucleotide sequences forthese genes are given in SEQ ID NO:1 (phaA), SEQ ID NO:4 (phaB) and SEQID NO:7 (phaC) where the nucleotide sequence does not include a signalsequence and, hence, the products, i.e. PhaA, PhaB and PhaC,respectively, are located in the cytosol.

Nucleotide sequences of phaA, phaB and phaC with a signal sequence todirect the products to the plastid are shown in SEQ ID NO:10, SEQ IDNO:13 and SEQ ID NO:16, respectively.

The nucleotide sequences encoding phaC1, phaG and phaJ may likewise comefrom any suitable source. In the case of phaC1, P. aeruginosa provides asuitable source. Nucleotide sequences encoding phaG and phaJ may bederived from, for example, Pseudomonas putida and Aeromonas caviae,respectively. The nucleotide sequence of phaC1 is given in SEQ ID NO:19,where the nucleotide sequence does not include a signal sequence and,hence, the product, i.e. PhaC1 is located in the cytosol.

Nucleotide sequences of phaC1, phaG and phaJ with a signal sequence todirect the products to the peroxisome and plastid (phaC1), and to theplastid (phaG) and peroxisome (phaJ), respectively, are shown in SEQ IDNO:22 and SEQ ID NO:25 and SEQ ID NO:28 and SEQ ID NO:31, respectively.

Clearly, the genetic sequences may be modified to insert any leader ortail sequence to direct the enzyme to an appropriate location in thecell.

Another aspect of the present invention contemplates a method forproducing PHAs in a C4 grass, said method comprising expressing one ormore genetic sequences comprising phaA, phaB, phaC, phaC1, phaG and/orphaJ or a derivative or homolog of any one of these in cells of a C4grass such that polymers of a PHA accumulate in the cytosol, storagevacuole, plastid or non-plastid organelle of said cell.

Where accumulation is in the cytosol, the PHA is preferably a PHB.

A homolog of a phaA, phaB, phaC, phaC1, phaG and phaJ includesnucleotide sequences having at least about 60% identity to one of SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:28 or SEQ IDNO:31 (or one of SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:22or SEQ ID NO:25) after optimal alignment or nucleotide sequences capableof hybridizing to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:19,SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16,SEQ ID NO: 22 or SEQ ID NO:25 or their complementary forms under lowstringency conditions.

Alternatively, or in addition, a homolog at the amino acid levelincludes an enzyme having an amino acid sequence with at least about 60%identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ IDNO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ IDNO:29 or SEQ ID NO:32.

Preferably, percentage similarities include at least about 70%, at leastabout 80%, at least about 90% and at least about 95% or above at thenucleotide and amino acid sequence levels such as 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99 and 100%.

Accordingly, reference herein to phaA, phaB, phaC phaC1, phaG, phaJ orPhaA, PhaB, PhaC, PhaC1, PhaG and PhaJ includes all homologs thereof.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces PHAs, said method comprisingintroducing into cells of said plant a genetic sequence comprising:—

-   -   (i) a nucleotide sequence encoding a phaA or homolog thereof;    -   (ii) a nucleotide sequence encoding phaB or homolog thereof;    -   (iii) a nucleotide sequence encoding phaC or homolog thereof;    -   (iv) a nucleotide sequence encoding phaC1 or homolog thereof;    -   (v) a nucleotide sequence encoding phaG or homolog thereof;    -   (vi) a nucleotide sequence encoding phaJ or homolog thereof    -   (vii) SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12        or a nucleotide sequence having at least 60% identity thereto        after optimal alignment, or capable of hybridizing to SEQ ID        NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12 or a        complementary form thereof under low stringency conditions;    -   (viii) SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID        NO:15 or a nucleotide sequence having at least 60% identity        thereto after optimal alignment, or capable of hybridizing to        SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or a        complementary form thereof under low stringency conditions;    -   (ix) SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18        or a nucleotide sequence having at least 60% identity thereto        after optimal alignment, or capable of hybridizing to SEQ ID        NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a        complementary form thereof under low stringency conditions;    -   (x) SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24        or SEQ ID NO:25 or SEQ ID NO:27 or a nucleotide sequence having        at least 60% identity thereto after optimal alignment, or        capable of hybridizing to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID        NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:27 or a        complementary form thereof under low stringency conditions;    -   (xi) SEQ ID NO:28 or SEQ ID NO:30 or a nucleotide sequence        having at least 60% identity thereto after optimal alignment, or        capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a        complementary form thereof under low stringency conditions;    -   (xii) SEQ ID NO:31 or SEQ ID NO:33 or a nucleotide sequence        having at least 60% identity thereto after optimal alignment, or        capable of hybridizing to SEQ ID NO:31 or SEQ ID NO:33 or a        complementary form thereof under low stringency conditions;        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, a sucrose-storing monocotyledonous plant. This aspect of thepresent invention includes progeny of the first generation plants.

A convenient C4 grass for use in the present invention is sugarcane.Sugarcane has certain advantages which make it a useful crop for use asa bioreactor including, inter alia, its efficient carbon fixation, highbiomass accumulation, rapid growth in subtropical and tropical climates,natural ability to accumulate large quantities of sucrose, hardiness,and ease of growth. In addition, a micropropagation system is alreadyavailable and an extensive industry infrastructure exists.

In order that PHAs may be produced in cells of a C4 grass, suitablesequences such as those derived from R. eutropha must be introduced intoand expressed in the cells. That is, the plant needs to undergo geneticmodification so that the metabolites and/or metabolic and/orbiosynthetic pathways can be harnassed for the production of the PHAs.This may conveniently be achieved through the use of genetic constructs,engineered to comprise nucleotide sequences required to effect PHAproduction.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is pHBA . . . .

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is vanillin.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) may be produced as aco-product with sucrose in sugarcane. A number of biological pathwayshave been discovered for the biosynthesis/biodegradation of vanillin. Atleast 2 of these have substrates which are available in plants, viz:

-   -   (i) 3-dehydroshikimic acid is a compound which is produced as an        intermediate in the shikimate pathway. A pathway has been        determined which converts this substrate via 3-dehydroshikimate        dehydratase to protocatechuic acid then to vanillic acid via        catechol-o-methyltransferase and finally to vanillin via aryl        aldehyde dehydrogenase.    -   (ii) Ferulic acid is a secondary metabolite of the        phenylpropanoid pathway involved in lignin synthesis. It is        converted in planta to feruloyl-CoA by feruloyl-CoA synthetase        which in turn is converted to vanillin by enoyl-CoA        hydratase/aldolase.

Accordingly, the present invention further contemplates a method forproducing vanillin in a C4 grass, said method comprising expressing oneor more genetic sequences which encode enzymes required for theproduction of vanillin, or a homolog or precursor thereof in a cell of aC4 grass such that the vanillin accumulates in the cytosol, storagevacuole, plastid or non-plastid organelle, or is extra-cellularlysecreted.

Either of these pathways, or a combination of these pathways, may beadapted to provide suitable genetic sequences for use in the productionof vanillin or precursors thereof in Sugarcane. Consequently, geneticsequences which “encode enzymes required for the production of vanillin”as used herein in the context of the present invention may comprise acombination of one or more of any sequence wherein the enzyme or enzymesthereby encoded usually operate in vivo singly or together to effect thebiosynthesis of vanillin or a precursor thereof.

Clearly, the genetic sequences may be modified to insert any leader ortail sequence to direct the enzyme to an appropriate location in thecell.

Another aspect of the present invention contemplates a method forproducing Vanillin in a C4 grass, said method comprising expressing oneor more genetic sequences encoding 3-dehydroshikimate dehydratase,catechol-o-methyltransferase, aryl aldehyde dehydrogenase, feruloyl-CoAsynthetase, enoyl-CoA hydratase and/or enoyl-CoA aldolase, in cells of aC4 grass such that vanillin accumulates anywhere in the cell orextra-cellular matrix of the plant.

Accordingly, reference herein to 3-dehydroshikimate dehydratase,catechol-o-methyltransferase, aryl aldehyde dehydrogenase, feruloyl-CoAsynthetase, enoyl-CoA hydratase and/or enoyl-CoA aldolase, includes allhomologs thereof.

In another embodiment, the present invention provides a method forgenerating a plant which produces vanillin or a precursor thereof, saidmethod comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a 3-dehydroshikimate        dehydratase and/or;    -   (ii) a nucleotide sequence encoding        catechol-o-methyltransferase;    -   (iii) a nucleotide sequence encoding aryl aldehyde        dehydrogenase;    -   (iv) a nucleotide sequence encoding feruloyl-CoA synthetase;    -   (v) a nucleotide sequence encoding enoyl-CoA hydratase;    -   (vi) a nucleotide sequence encoding enoyl-CoA aldolase;    -   (vii) a nucleotide sequence encoding a homolog of any one of (i)        though (vi)        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane. Included in this aspect of the present inventionare progeny of the first generation plants.

In order that Vanillin may be produced in cells of a C4 grass, suitablesequences such as those encoding one or more Vanillin biosyntheticenzymes must be introduced into and expressed in the cells. That is, theplant needs to undergo genetic modification so that the metabolitesand/or metabolic and/or biosynthetic pathways can be harnessed for theproduction of the vanillin or a precursor thereof. This may convenientlybe achieved through the use of genetic constructs, engineered tocomprise nucleotide sequences required to effect vanillin production.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is sorbitol.

Sorbitol is a polyol that is found naturally in many fruits. To satisfythe high demand for this compound it is synthesized industrially byhydrogenation of corn-derived glucose in aqueous solution usingnickel-containing catalysts. The majority of the sorbitol produced isconsumed in the manufacture of toothpaste, confectionary, and ascorbicacid.

A new market for sorbitol in the polymer sector has been described byindustry. Isosorbide or 1,4-3,6-dianhydrosorbitol is produced by theacid catalyzed dehydration of sorbitol. Recent patents have demonstratedthat copolymers of isosorbide and polyethylene terephthalate (PET)exhibit superior strength and rigidity compared to PET alone. 4.4billion lb of PET is currently used in food and beverage containers(Source: US Dept. Energy, 2001). Replacing PET with the isosorbide-PETcopolymer (sPET) would reduce the overall consumption ofpetroleum-derived PET because less sPET is needed to achieve theequivalent strength. The projected sPET production is 1 billion lb peryear by 2020, utilizing 100 million lb of isosorbide (Source: US Dept.Energy, 2001).

Sorbitol is already produced from a renewable feedstock. The mainincentive to use sugarcane as a sorbitol biofactory is to capitalizeupon a potential future demand for this product by the plasticsindustry.

Sorbitol can also be converted to other useful chemicals. Propyleneglycol, ethylene glycol, and glycerol can be derived from catalytichydrogenolysis of sorbitol. These chemical feedstocks are currentlyderived from petrochemicals.

The biosynthesis of sorbitol produces the coproduct gluconolactone. Theenzyme glucono-δ-lactonase can convert the gluconolactone into gluconicacid. Gluconic acid is used as a food acidulant, antioxidant, and aclarifier in wines and softdrinks.

Zymomonas mobilis is able to produce sorbitol from sucrose or a mixtureof glucose and fructose in a one-step reaction catalysed by theglucose-fructose oxidoreductase GFOR (Genbank accession no. Z80356,M97379). The glucose is oxidized to gluconolactone while the fructose isreduced to sorbitol.

glucose+fructose→sorbitol+gluconolactone

Without limiting the present invention to any one method or mode ofaction, sorbitol production in sugarcane could be achieved by usingGFOR. This involves constructing an expression cassette by fusing GFORto the maize polyubiquitin promoter and nopaline synthase terminator andintroducing the cassette into sugarcane callus by biolistictransformaton. The Z. mobilis GFOR is not membrane-bound and resides inthe periplasm and should work equally well as a cytosolic enzyme insugarcane.

Sorbitol production is unlikely to be toxic in sugarcane since sorbitolis found in numerous fruits (apples, pears, plums, berries, cherries).Sorbitol functions physiologically to regulate osmotic stress henceextremely high levels may be detrimental and vacuolar storage maycircumvent this problem.

Accordingly, the present invention further contemplates a method forproducing sorbitol in a C4 grass, said method comprising expressing oneor more genetic sequences which encode enzymes required for theproduction of sorbitol, or a homolog or precursor thereof in a cell of aC4 grass such that the sorbitol accumulates in the cytosol, storagevacuole, plastid or non-plastid organelle, or is secretedextra-cellularly.

In addition to the glucose-fructose oxidoreductase pathway, othernucleotide sequences may encode other enzymes suitable for use in theproduction of sorbitol or precursors thereof in Sugarcane. Consequently,genetic sequences which “encode enzymes required for the production ofsorbitol” as used herein in the context of the present invention maycomprise a combination of one or more of any sequence wherein the enzymeor enzymes thereby encoded usually operate in vivo singly or together toeffect the biosynthesis of sorbitol or a precursor thereof.

Clearly, the genetic sequences may be modified to insert any leader ortail sequence to direct the enzyme to an appropriate location in thecell.

Another aspect of the present invention contemplates a method forproducing sorbitol in a C4 grass, said method comprising expressing oneor more genetic sequences encoding a glucose-fructose oxidoreductase orhomolog thereof, in cells of a C4 grass such that sorbitol accumulatesanywhere in the cell or extra-cellular matrix of the plant.

In a preferred embodiment, the glucose-fructose oxidoreductase is thatencoded by the nucleic acid sequence set forth in Genbank accessionnumber Z80356 or M97379, or a nucleotide sequence having at least 60%identity thereto after optimal alignment, or capable of hybridizing tothese sequences under low stringency conditions.

Accordingly, reference herein to glucose-fructose oxidoreductase,includes all homologs thereof.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces sorbitol or a precursor thereof, saidmethod comprising introducing into cells of said plant a geneticsequence comprising a nucleotide sequence encoding a glucose-fructoseoxidoreductase or homolog thereof and then regenerating a plant fromsaid cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that sorbitol may be produced in cells of a C4 grass, suitablesequences such as those encoding one or more sorbitol biosyntheticenzymes must be introduced into and expressed in the cells. That is, theplant needs to undergo genetic modification so that the metabolitesand/or metabolic and/or biosynthetic pathways can be harnessed for theproduction of the sorbitol or a precursor thereof. This may convenientlybe achieved through the use of genetic constructs, engineered tocomprise nucleotide sequences required to effect sorbitol production.

Clearly, the genetic sequences may be modified to insert any leader,tail or signal sequences to direct the enzyme to an appropriate locationin the cell.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is indigo.

Until the end of the 19th century, the sole source of indigo was fromplants, woad (Isatis tinctoria) and Dyer's Knotweed (Polygonumtinctorum) in temperate climates and Indigofera species in the tropics.Woad was widely grown in Europe, making some regions, especiallyToulouse (France) and Erfurt (Germany), very wealthy until the end ofthe 16th century.

Plant-based indigo was almost entirely replaced in the 20th century bysynthetic indigo. Today indigo is still regarded as a high valuespecialty chemical used mainly as a dye in the textile industry. It isproduced synthetically from naphthalene by the Heumann synthesisreaction.

The chief incentive to use sugarcane as an indigo biofactory is toprovide a manufacturing route that will produce relatively inexpensiveindigo from a renewable feedstock.

Indigo production by microbial fermentation has been demonstrated byexpressing the genes that mediate indigo formation in E. coli. Thepigment is derived by converting endogenous tryptophan to indole usingthe Enterobacter aerogenes tryptophanase or L-tryptophan indole lyase EC4.1.99.1 (Genbank accession no. D14297). Subsequently the indole isconverted to indigo via two possible reactions.

Route A: Pseudomonas putida napthalene dioxygenase (Genbank accessionno. M83949)

Route B: Ralstonia eutropha bec gene (Genbank accession no. AF306552)

These pathways are graphically depicted in FIG. 8.

Without limiting the present invention to any one method or mode ofaction, indigo production in sugarcane involves constructing anexpression cassette by fusing the aforementioned genes to the maizepolyubiquitin promoter and nopaline synthase terminator and introducingthe cassette into sugarcane callus by biolistic transformaton.Tryptophan is a product of the plant shikimate pathway, which isresponsible for synthesizing lignin precursors. The cloned genes may beplastid-targeted since the shikimate pathway reactions reside in thiscompartment. The available metabolic flux in this pathway is expected tobe significant.

Aeration of the sugarcane juice will lead to spontaneous oxidation ofindoxyl to an insoluble indigo precipitate. The solid precipitate shouldbe easy to separate from the solution by filtration or centrifugation.

Another aspect of the present invention contemplates a method forproducing indigo in a C4 grass, said method comprising expressing one ormore genetic sequences encoding tryptophanase, L-tryptophan indolelyase, napthalene dioxygenase, and/or the Ralstonia eutropha bec gene,or homolgs thereof, in cells of a C4 grass such that indigo accumulatesanywhere in the cell or extracellular matrix of the plant.

In a preferred embodiment, indigo accumulates in the plastid of theplant cell.

Accordingly, reference herein to genetic sequences encodingtryptophanase, L-tryptophan indole lyase, napthalene dioxygenase, and/orthe Ralstonia eutropha bec gene includes all homologs thereof.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces indigo or a precursor thereof, saidmethod comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding genetic sequences encoding        tryptophanase;    -   (ii) a nucleotide sequence encoding L-tryptophan indole lyase;    -   (iii) a nucleotide sequence encoding napthalene dioxygenase;    -   (iv) a nucleotide sequence comprising the Ralstonia eutropha bec        gene;    -   (v) the nucleotide sequence set forth in Genbank accession        number D14279, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank D14279 under low stringency conditions.    -   (vi) the nucleotide sequence set forth in Genbank accession        number M83949, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank M83949 under low stringency conditions.    -   (vii) the nucleotide sequence set forth in Genbank accession        number AF306552, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank AF306552 under low stringency conditions.        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that indigo may be produced in cells of a C4 grass, suitablesequences such as those encoding one or more indigo biosynthetic enzymesmust be introduced into and expressed in the cells. That is, the plantneeds to undergo genetic modification so that the metabolites and/ormetabolic and/or biosynthetic pathways can be harnessed for theproduction of the indigo or a precursor thereof. This may convenientlybe achieved through the use of genetic constructs, engineered tocomprise nucleotide sequences required to effect indigo production.

Clearly, the genetic sequences may be modified to insert any leader ortail sequence to direct the enzyme to an appropriate location in thecell. In a preferred embodiment the leader, tail or signal sequencedirects the indigo biosynthetic enzyme to be localized in the plastid.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is a mixture ofdifferent chain length polymers of fructose monomers, such as fructans.

Fructan, or levan as it is often called, is a fructosehomopolysaccharide that is linked to a terminal glucose residue.Fructans are a storage carbohydrate in some plants such as Jerusalemartichoke and chicory. Certain bacilli can also synthesize fructans.

Despite a plethora of potential applications, this polymer is not yetwidely used. Some of the possible uses cited by the literature are:

-   -   (i) Low calorie sweetener. Fructans possess a sweet taste but        cannot be degraded in humans.    -   (ii) Dietary fibre    -   (iii) Bulking agent    -   (iv) Raw material for biodegradable plastics, detergents, and        adhesives

Fructans may also be an inexpensive source of fructose in the future.The food industry is rapidly adopting fructose as the preferredsweetener over sucrose. Fructose may be up to 1.8 times sweeter thansucrose. Consequently, less fructose is needed to derive the sameeffect. Fructose syrup is presently obtained by hydrolysis of starch toglucose followed by enzymatic isomerization of glucose to fructose. Theresultant solution is an equilibrium mixture of glucose/fructose thatmust be further purified by ion chromatography to obtain near purefructose. This final step purportedly adds significantly to the cost offructose manufacture. It would be possible to avoid this step if thestarting material contained only fructose. Simple hydrolysis of fructanswill yield pure fructose at a reduced cost compared with using starch asthe raw material.

Incentives to use sugarcane as fructan biofactory include:

-   -   (i) To create a market for this product. A demand for fructans        would develop if sufficient amounts were made available. The        disadvantage of the existing fructan flora is the low        harvestable weight per plant.    -   (ii) To provide an alternative and inexpensive route for        fructose production.    -   (iii) In subtropical and tropical climates sugarcane exhibits        fast growth and very high biomass yields. The high rate of CO₂        fixation due to C4 photosynthesis should facilitate a rapid        accumulation of fructans.    -   (iv) Vegetative propagation ensures a stable germplasm and hence        predictable product yields.

Naturally occurring fructans may contain 10 to 100,000 fructoseresidues. Bacteria produce the larger fructans whilst those occurring inplants are smaller. The larger polymers are desirable because they areless soluble in water and consequently easier to extract. Largerfructans will not affect the osmotic pressure in the cell to the samedegree as smaller molecules. Therefore it is possible to store greaterquantities of fructan before the cell is affected.

Numerous bacterial fructosyltransferases or levansucrases have beencharacterized such as Genbank accession no. AY150365, from Bacillussubtilis. These enzymes catalyze the transfer of the D-fructosyl residuefrom sucrose to the β-2,6-linked residues of fructan.

Sucrose→fructan+glucose

Without limiting the present invention to any one method or mode ofaction, fructan production in sugarcane would be achieved byconstructing an expression cassette containing levansucrase, the maizepolyubiquitin promoter and nopaline synthase terminator and introducingthe cassette into sugarcane callus by biolistic transformaton.

Another aspect of the present invention contemplates a method forproducing fructans in a C4 grass, said method comprising expressing oneor more genetic sequences encoding a bacterial fructosyltransferase orlevansucrase in cells of a C4 grass such that a fructan accumulatesanywhere in the cell or extra-cellular matrix of the plant.

In a preferred embodiment, fructan accumulates in the apoplast orvacuole of the plant cell.

Accordingly, reference herein to genetic sequences encodingfructosyltransferases and levansucreases includes all homologs thereof.

In another embodiment, the present invention relates to a method forgenerating a C4 grass plant which produces a fructan or a precursorthereof, said method comprising introducing into cells of said plant agenetic sequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a fructosyltransferase or        homolog thereof;    -   (ii) a nucleotide sequence encoding a levansucrase or homolg        thereof;    -   (iii) the nucleotide sequence set forth in Genbank accession        number AY150365, or a nucleotide sequence having at least 60%        identity thereto after optimal alignment, or capable of        hybridizing to Genbank AY150365 under low stringency conditions.        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that a fructan may be produced in cells of a C4 grass, suitablesequences such as those encoding one or more indigo biosynthetic enzymesmust be introduced into and expressed in the cells. That is, the plantneeds to undergo genetic modification so that the metabolites and/ormetabolic and/or biosynthetic pathways can be harnessed for theproduction of the indigo or a precursor thereof. This may convenientlybe achieved through the use of genetic constructs, engineered tocomprise nucleotide sequences required to effect fructan production.

Clearly, the genetic sequences may be modified to insert any leader,tail or signal sequence to direct the enzyme to an appropriate locationin the cell. In a preferred embodiment, to maximise fructan productionin sugarcane, levansucrase is directed to the apoplast or vacuole tomaximize access to substrate for conversion.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is lactic acid(2-Hydroxypropanoic acid).

The world market for solvent replacement, biodegradable plastics andoxygenated chemicals derived from lactic acid exceeds US$ 10 billion(Argonne National Laboratory, US DOE).

Without limiting the present invention to any one method or mode ofaction, lactic acid production in sugarcane involves the followinggeneral steps:

-   -   (i) Obtain or clone lactate dehydrogenase (LDH) or a homolog        thereof:    -   (ii) Express gene in sugarcane (cytosol, therefore no targeting        is required)    -   (iii) Regenerate plants and evaluate for lactate (or derivative)        production

Traditionally, lactic acid purification has been a complex chemicalprocess. However, recent advances have simplified this process and madeit significantly cheaper. It is anticipated that lactic acid can beremoved from the post-crushing millstream without great difficulty orextensive modification of existing structures.

Accordingly, the present invention further contemplates a method forproducing lactic acid in a C4 grass, said method comprising expressingone or more genetic sequences which encode enzymes required for theproduction of lactic acid, or a homolog or precursor thereof in a cellof a C4 grass such that the lactic acid accumulates in the cytosol,storage vacuole, plastid or non-plastid organelle, or is secretedextra-cellularly.

Genetic sequences which “encode enzymes required for the production oflactic acid” as used herein in the context of the present invention maycomprise a combination of one or more of any sequence wherein the enzymeor enzymes thereby encoded usually operate in vivo singly or together toeffect the biosynthesis of lactic acid or a precursor thereof.

Clearly, the genetic sequences may be modified to insert any leader ortail sequence to direct the enzyme to an appropriate location in thecell.

In a preferred embodiment the lactate dehydrogenase nucleic acidsequence is expressed without a signal sequence such that the enzyme isactive in the cytosol.

Another aspect of the present invention contemplates a method forproducing lactic acid in a C4 grass, said method comprising expressingone or more genetic sequences encoding lactate dehydrogenase or ahomolog thereof in cells of a C4 grass such that lactic acid accumulatesanywhere in the cell or extracellular matrix of the plant.

Accordingly, reference herein to lactate dehydrogenase, includes allhomologs thereof.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces lactic acid or a precursor thereof,said method comprising introducing into cells of said plant a geneticsequence encoding lactate dehydrogenase or homolog thereof, and thenregenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that lactic acid may be produced in cells of a C4 grass,suitable sequences such as those encoding one or more lactic acidbiosynthetic enzymes must be introduced into and expressed in the cells.That is, the plant needs to undergo genetic modification so that themetabolites and/or metabolic and/or biosynthetic pathways can beharnessed for the production of the lactic acid or a precursor thereof.This may conveniently be achieved through the use of genetic constructs,engineered to comprise nucleotide sequences required to effect lacticacid production.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is adipic acid.

Adipic acid is classified as a bulk chemical and is among the top fiftychemicals produced in the US. It is principally used in the productionof nylon 66, polyeurethane resins and plasticizers. Nearly 90% is usedto produce nylon-6,6, a synthetic polymer developed by DuPont in the1930's. This polyamide is formed by the condensation of adipic acid and1,6-diaminohexane.

Adipic acid is presently produced industrially by benzene-basedsynthetic chemistry. Catalytic hydrogenation of benzene followed by airoxidation yields a ketone/alcohol mixture (cyclohexanone/cyclohexanol)that is further oxidized with nitric acid to produce adipic acid.

Incentives to use sugarcane as an adipic acid biofactory include:

-   -   (i) Provide a renewable feedstock for adipic acid manufacture.    -   (ii) Eliminate the production of the toxic nitrous oxide        byproduct that accompanies traditional adipic acid synthesis.    -   (iii) Capitalize upon the high demand for this product.    -   (iv) Access to low cost molasses to produce products by        fermentation.    -   (v) Access to low operating and infrastructure costs through        co-location of an extraction facility (or fermentation facility)        with a sugar mill.    -   (vi) In subtropical and tropical climated sugarcane exhibits        fast growth and very high biomass yields. This is a prerequisite        for economical bulk chemical production.    -   (vii) Vegetative propagation ensures a stable germplasm and        hence predictable product yields.

Without limiting the present invention to any one method or mode ofaction, adipic acid may be produced in sugarcane by one of twoapproaches.

I. Synthesis from Cis, Cis-Muconic Acid

Niu et al. (Biotechnol. Prog., 18: 201-211, 2002) describe amicrobiological route for the production of adipic acid using E. coli.Three genes were introduced into E. coli to produce cis, cis-muconicacid that was subsequently purified from the fermentation broth andconverted to adipic acid by catalytic hydrogenation (step g, 10% Pt/C,H₂, 3400 kPa, 25° C.). This final step has a 97% conversion efficiency.

The synthesis of cis, cis-muconic acid in sugarcane involves making useof the shikimate pathway. In order to use the shikimate pathway toproduce cis, cis-muconic acid the following biosynthetic enzymes, orhomologs thereof are introduced into sugarcane:

Klebsiella pneumoniae 3-dehydroshikimate dehydratase (aroZ)-enzyme d

3-dehydroshikimate→protocatechuate

Klebsiella pneumoniae protocatechuate decarboxylase (aroY)-enzyme e

Protocatechuate→catechol

Acinetobacter calcoaceticus catechol 1,2-dioxygenase (catA)-enzyme f

Catechol+O₂→cis, cis-muconic acid

Introduction of these genes into sugarcane involves constructing anexpression cassette by fusing the genes described above to the maizepolyubiquitin promoter and nopaline synthase terminator and introducingthe cassette into sugarcane callus by biolistic transformation. Catecholis probably produced in most plants, and therefore, it may beunnecessary to clone additional copies of 3-dehydroshikimate dehydrataseor protocatechuate decarboxylase. Preferrably, the cloned gene(s) areplastid-targeted since the shikimate pathway reactions reside in thiscompartment.

The merits of using plant secondary metabolism to synthesize interestingproducts have often been promoted in the literature (Verpoorte andMemelink, Curr. Opin. Biotech. 13: 181-187, 2002). The shikimate pathwayexecutes a central role in plant secondary metabolism. This is one ofthe most active pathways in plants in terms of carbon flux owing to thefact that it is the source of lignin precursors. This makes it anattractive candidate for metabolic engineering.

II. Synthesis from Petroselinic Acid

Bio-based adipic acid can be obtained through ozonolysis (O₃) ofpetroselinic acid (18:1 Δ^(6 cis)), as depicted in FIG. 10. Thecoproduct lauric acid is also a potential source of feedstock fordetergent manufacture.

The seed oil of the coriander spice plant contains 80-90% petroselinicacid. A 36 kDa putative acyl-ACP desaturase (Genbank accession no.M93115) has been identified from coriander seed extracts and thecorresponding cDNA was able to confer the ability to producepetroselinic acid in tobacco callus (Cahoon et al. Proc. Natl. Acad.Sci. USA 89: 11184-11188, 1992).

The metabolic pathway for producing petroselinic acid is unclear,however, evidence suggests that it is formed by the desaturation ofpalmitoyl-ACP by the 36 kDa desaturase followed by elongation to formpetroselinic acid (Cahoon and Ohlrogge, Plant Physiol., 104: 827-837,1994).

16:0-ACP→16:1Δ⁴-ACP→18:1Δ⁶-ACP

Recent studies have identified a 3-ketoacyl-ACP synthase (Genbankaccession no. AF263992) associated with the two-carbon elongation of16:1 Δ⁴-ACP (Mekhedov et al., Plant Mol. Biol. 47: 507-518, 2001).

Cis, cis-muconic acid in sugarcane juice would be converted to adipicacid by catalytic hydrogenation. The adipic acid in the resultantsolution can be recovered by solvent extraction. The solution iscontacted with chloroform or methylene chloride and the adipic acidrecovered in the aqueous fraction. The aqueous fraction would then beevaporated to yield crystalline adipic acid.

Accordingly, the present invention further contemplates a method forproducing adipic acid, or a precursor thereof such as cis, cis-muconicacid, in a C4 grass, said method comprising expressing one or moregenetic sequences which encode enzymes required for the production ofadipic acid and/or cis, cis-muconic acid, or a homolog or precursorthereof in a cell of a C4 grass such that adipic acid and/or cis,cis-muconic acid accumulates in the cytosol, storage vacuole, plastid ornon-plastid organelle, or is secreted extra-cellularly.

Either of the hereinbefore described pathways for the production of cis,cis-muconic acid, or a combination of these pathways, may be adapted toprovide suitable genetic sequences for use in the production of vanillinor precursors thereof in Sugarcane. Consequently, genetic sequenceswhich “encode enzymes required for the production of cis, cis-muconicacid” as used herein in the context of the present invention maycomprise a combination of one or more of any sequence wherein the enzymeor enzymes thereby encoded usually operate in vivo singly or together toeffect the biosynthesis of cis, cis-muconic acid or a precursor thereof.

Clearly, the genetic sequences may be modified to insert any leader,tail or signal sequence to direct the enzyme to an appropriate locationin the cell. Preferrably the leader tail or signal sequences lead to theco-localization of the adipic acid biosynthetic enzymes with theendogenous skimimate pathway enzymes in the plant. More preferably, saidenzymes are localized in the plastid.

Another aspect of the present invention contemplates a method forproducing cis, cis-muconic acid or adipic acid in a C4 grass, saidmethod comprising expressing one or more genetic sequences encoding3-dehydroshikimate dehydratase, protochatechuate decarboxylase, catechol1,2-dioxygenase and/or 3-ketoacyl-ACP synthase in cells of a C4 grasssuch that adipic acid or an adipic acid precursor accumulates anywherein the cell or extracellular matrix of the plant.

Accordingly, reference herein to 3-dehydroshikimate dehydratase,protochatechuate decarboxylase, catechol 1,2-dioxygenase and/or3-ketoacyl-ACP synthase, includes all homologs thereof.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces adipic acid or a precursor thereof,said method comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a 3-dehydroshikimate        dehydratase and/or;    -   (ii) a nucleotide sequence encoding protochatechuate        decarboxylase;    -   (iii) a nucleotide sequence encoding catechol 1,2-dioxygenase;    -   (iv) a nucleotide sequence encoding 3-ketoacyl-ACP synthase;        and/or    -   (v) a nucleotide sequence encoding a homolog of any one of (i)        through (iv).        and then regenerating a plant from said cells.

In a preferred embodiments:

-   -   (i) the nucleotide sequence encoding a 3-dehydroshikimate        dehydatase is the aroZ gene from Klebsiella pneumoniae, or a        homolog thereof;    -   (ii) the nucleotide sequence encoding a protochatechuate        decarboxylase is the aroY gene from Klebsiella pneumoniae, or a        homolog thereof;    -   (iii) the nucleotide sequence encoding a 1,2-dioxygenase is the        catA gene from Acinetobacter calcoaceticus, or a homolog        thereof;    -   (iv) the nucleotide sequence encoding a 3-ketoacyl-ACP synthase        is the nucleotide sequence set forth in Genbank Accession number        AF263992, or a nucleotide sequence having at least 60% identity        thereto after optimal alignment, or capable of hybridizing to        Genbank AF263992 under low stringency conditions.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that adipic acid or a precursor thereof may be produced incells of a C4 grass, suitable sequences such as those encoding one ormore adipic acid biosynthetic enzymes must be introduced into andexpressed in the cells. That is, the plant needs to undergo geneticmodification so that the metabolites and/or metabolic and/orbiosynthetic pathways can be harnessed for the production of adipic acidor a precursor thereof. This may conveniently be achieved through theuse of genetic constructs, engineered to comprise nucleotide sequencesrequired to effect the production of adipic acid and/or precursorsthereof.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is 1,3-propanediol(1,3-PD).

1,3-PD is a bifunctional alcohol that can be used as a monomer innumerous polycondensation reactions to produce polyesters,polyurethanes, and polyethers. The high cost of chemical synthesis,reportedly US$30/kg (Biebl et al., Appl. Microbiol. Biotechnol., 52:289-297, 1999) has restricted its use in the past to specialty marketssuch as dioxane production and the solvent market.

1,3-PD is synthesized using a process in which ethylene oxide is reactedwith carbon dioxide and hydrogen. An alternative method, the Degussaprocess, is based upon hydrolysis of acrolein followed by catalytichydrogenation. Both routes involve the use of petrochemical feedstock.

1,3-PD is a natural product of glycerol fermentation in a fewenterobacteria and clostridia.

Incentives to use sugarcane as 1,3-PD biofactory include:

-   -   (i) Provide a renewable feedstock for 1,3-PD manufacture.    -   (ii) Provide a high volume, low cost source of 1,3-PD to        facilitate expansion of the market.    -   (iii) Capitalize upon the high demand for this product.    -   (iv) Access to low cost molasses to produce products by        fermentation.    -   (v) Access to low operating and infrastructure costs through        co-location of an extraction facility (or fermentation facility)        with a sugar mill.    -   (vi) In subtropical and tropical climated sugarcane exhibits        fast growth and very high biomass yields. This is a prerequisite        for economical bulk chemical production.    -   (vii) Vegetative propagation ensures a stable germplasm and        hence predictable product yields.

The metabolic reactions that convert glycerol to 1,3-PD have beenestablished from Klebsiella pneumoniae:

Klebsiella pneumoniae glycerol dehydratase (dhaB)

glycerol→3-hydroxypropionaldehyde+H₂O

Klebsiella pneumoniae 1,3-propanediol oxidoreductase (dhaT)

3-hydroxypropionaldehyde+NADH→1,3-propanediol+NAD

Sugarcane does not naturally produce glycerol therefore the reactionsthat convert triose phosphates to glycerol must also be engineered intosugarcane.

Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase

dihydroxyacetone phosphate+NADH→glycerol-3-phosphate+NAD

Saccharomyces cerevisiae glycerol-3-phosphatase

glycerol-3-phosphate+ADP→glycerol+ATP

Without limiting the present invention to any one method or mode ofaction, all four new genes are cloned into sugarcane to convert it intoa 1,3-PD biofactory. These genes are assembled into an expressioncassette containing the maize polyubiquitin promoter and nopalinesynthase terminator. The cassette is introduced into sugarcane callus bybiolistic transformation and expression will be targeted to the cytosol.The accumulation of 1,3-PD in plant tissue will be assayed from plantextracts by conventional HPLC.

1,3-PD can be recovered from sugarcane juice by extraction withcyclohexane followed by vaporization of the residual solvent.Alternatively, distillation may be employed. Use of cyclohexane isenvironmentally unsound and distillation is energy intensive.Consequently, a method has been patented that describes the use of ionexclusion resins to recover 1,3-PD (WO0173097 Method of recovering1,3-propanediol from fermentation broth, Archer Daniels Midland Co.,2001).

Accordingly, the present invention further contemplates a method forproducing 1,3-propanediol in a C4 grass, said method comprisingexpressing one or more genetic sequences which encode enzymes requiredfor the production of 1,3-propanediol, or a homolog or precursor thereofin a cell of a C4 grass such that the 1,3-propanediol accumulates in thecytosol, storage vacuole, plastid or non-plastid organelle, or issecreted extra-cellularly.

Any of the disclosed biosynthetic steps, or a combination of these, maybe adapted to provide suitable genetic sequences for use in theproduction of 1,3-propanediol, or precursors thereof, in sugarcane.Consequently, genetic sequences which “encode enzymes required for theproduction of 1,3-propanediol” as used herein in the context of thepresent invention may comprise a combination of one or more of anysequence wherein the enzyme or enzymes thereby encoded usually operatein vivo singly or together to effect the biosynthesis of 1,3-propanediolor a precursor thereof.

Clearly, the genetic sequences may be modified to insert any leader ortail sequence to direct the enzyme to an appropriate location in thecell.

The present invention contemplates a method for producing1,3-propanediol in a C4 grass, the method comprising expressing one ormore genetic sequences encoding glycerol dehydratase, 1,3-propanedioloxidoreductase, glycerol-3-phosphate dehydrogenase andglycerol-3-phosphatase in cells of a C4 grass such that 1,3-propanediolaccumulates anywhere in the cell or extra-cellular matrix of the plant.

Accordingly, reference herein to glycerol dehydratase, 1,3-propanedioloxidoreductase, glycerol-3-phosphate dehydrogenase andglycerol-3-phosphatase includes all homologs thereof.

In another aspect, the present invention contemplates a method forgenerating a plant which produces 1,3-propanediol or a precursorthereof, said method comprising introducing into cells of said plant agenetic sequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a glycerol dehydratase        and/or;    -   (ii) a nucleotide sequence comprising the dhaB gene from        Klebsiella pneumoniae, or a homolg thereof;    -   (iii) a nucleotide sequence encoding 1,3-propanediol        oxidoreductase;    -   (iv) a nucleotide sequence comprising the dhaT gene from        Klebsiella pneumoniae or homolg thereof    -   (v) a nucleotide sequence encoding glycerol-3-phosphate        dehydrogenase;    -   (vi) a nucleotide sequence encoding glycerol-3-phosphatase;        and/or    -   (vii) a nucleotide sequence encoding a homolog of any one of (i)        through (iv)        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that 1,3-propanediol may be produced in cells of a C4 grass,suitable sequences such as those encoding one or more 1,3-propanediolbiosynthetic enzymes must be introduced into and expressed in the cells.That is, the plant needs to undergo genetic modification so that themetabolites and/or metabolic and/or biosynthetic pathways can beharnessed for the production of the 1,3-propanediol or a precursorthereof. This may conveniently be achieved through the use of geneticconstructs, engineered to comprise nucleotide sequences required toeffect 1,3-propanediol production.

Clearly, the genetic sequences may be modified to insert any leader,tail or signal sequence to direct the enzyme to an appropriate locationin the cell.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is 2-phenylethanol(2-PE).

2-phenylethanol (2-PE) is an important flavour and fragrance compoundwith a rose-like odour. Most of the world's annual production of severalthousand tons is synthesised by chemical means but, due to increasingdemand for natural flavours, alternative production methods are beingsought (Etschmann et al. Appl Micribiol Biotechnol 59:1-8, 2002)

A biological pathway for the biosynthesis of 2-PE is presented in FIG.11.

Sugarcane has a productive phenylpropanoid pathway and should adaptreadily to increased demands placed on it for synthesis of 2-PE.

Accordingly, in another aspect, the present invention furthercontemplates a method for producing 2-phenylethanol in a C4 grass, saidmethod comprising expressing one or more genetic sequences which encodeenzymes required for the production of 2-phenylethanol, or a homolog orprecursor thereof in a cell of a C4 grass such that the 2-phenylethanolaccumulates in the cytosol, storage vacuole, plastid or non-plastidorganelle, or is secreted extra-cellularly.

Any of the disclosed biosynthetic steps, or a combination of these, maybe adapted to provide suitable genetic sequences for use in theproduction of 2-phenylethanol, or precursors thereof, a C4 grass such asin sugarcane. Consequently, genetic sequences which “encode enzymesrequired for the production of 2-phenylethanol” as used herein in thecontext of the present invention may comprise a combination of one ormore of any sequence wherein the enzyme or enzymes thereby encodedusually operate in vivo singly or together to effect the biosynthesis of2-phenylethanol or a precursor thereof.

Another aspect of the present invention contemplates a method forproducing 2-phenylethanol in a C4 grass, said method comprisingexpressing one or more genetic sequences encoding aromatic-L-amino aciddecarboxylase, 2-phenylethylamine oxidase and aryl-alcohol dehydrogenasein cells of a C4 grass such that 2-phenylethanol accumulates anywhere inthe cell or extracellular matrix of the plant.

Accordingly, reference herein to aromatic-L-amino acid decarboxylase,2-phenylethylamine oxidase and aryl-alcohol dehydrogenase includes allhomologs thereof.

In another embodiment, the present invention contemplates a method forgenerating a plant which produces 2-phenylethanol or a precursorthereof, said method comprising introducing into cells of said plant agenetic sequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding a aromatic-L-amino acid        decarboxylase and/or;    -   (ii) a nucleotide sequence encoding 2-phenylethylamine oxidase;    -   (iii) a nucleotide sequence encoding aryl-alcohol dehydrogenase;        and/or    -   (iv) a nucleotide sequence encoding a homolog of any one of (i)        through (iii)        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that 2-phenylethanol may be produced in cells of a C4 grass,suitable sequences such as those encoding one or more 2-phenylethanolbiosynthetic enzymes must be introduced into and expressed in the cells.That is, the plant needs to undergo genetic modification so that themetabolites and/or metabolic and/or biosynthetic pathways can beharnessed for the production of the 2-phenylethanol or a precursorthereof. This may conveniently be achieved through the use of geneticconstructs, engineered to comprise nucleotide sequences required toeffect 2-phenylethanol production.

Clearly, the genetic sequences may be modified to insert any leader,tail or signal sequence to direct the enzyme to an appropriate locationin the cell.

In another preferred embodiment of the present invention, the plantselected is a C4 grass and the product of interest is pHBA.

A schematic depicting the pHBA biosynthetic pathway is shown in FIG. 14.

In order to effect pHBA production in sugarcane, a chloroplast-targetedversion of E. coli situated between the maize ubi-1 promoter and nosterminator of the expression construct pU3z-mcs-nos, was co-bombardedwith a plasmid containing a selectable marker (pUKN) into embryogenicsugarcane callus to yield the UC series of transgenic lines. The UHseries of plants was generated in the same manner using an analogousexpression construct that contained the ORF of the P. fluorescens HCHLgene. The regenerated plants were grown in a greenhouse for four weeksand were then analyzed for pHBA accumulation in leaf tissue using HPLC.

pHBA accumulated in the transformed plants as two glucose conjugates,ie, a phenolic glucoside and a glucose ester. Both compounds contained asingle glucose molecule that was attached by a 1-O— -D linkage to thehydroxyl or carboxyl group of pHBA. The predominant product in all ofthe plants examined was the phenolic glucoside, which accounted for atleast 90% of the pHBA.

The mean value for the population was 0.41%±0.04% of dry weight (DW),which is almost 30-fold higher than the mean value for thenon-transgenic control plants 0.014%±0.01% DW. More important, the pHBAglucoside content of the best plant was 1.5% DW, which is equivalent to0.69% DW free pHBA after correcting for the attached glucose molecule.This value is three times higher than the highest value obtained withtransgenic tobacco plants expressing a different chloroplast-targetedversion of CPL. The HCHL-expressing sugarcane plants accumulated evenhigher levels of pHBA. The mean value for total pHBA glucose conjugatesin the UH lines was 0.70%±0.07% DW, and the highest level observed atthis stage of development was 2.6% DW.

Based on the results obtained with the 4-week-old plants, a subset ofthe primary transformants was selected for further evaluation, and leaflevels of pHBA were determined after 16 weeks additional growth.Included in this analysis were the two CPL-expressing plants thatpreviously exhibited the highest levels of product accumulation (UC63and UC65) and five HCHL-expressing plants. The methanol-extractedsamples were subjected to acid hydrolysis, which quantitativelyhydrolyzes both pHBA glucose conjugates, and free pHBA was determined byHPLC.

It was anticipated that pHBA production would continue throughoutdevelopment and that the 20-week-old plants would have higher levels ofpHBA glucosides than the 4-week-old plants. However, the increase inpHBA content with age was not very dramatic nor was it universallyobserved when product accumulation was expressed on a dry weight basis(FIG. 3A). Part of the explanation for this is the lower water contentof the older plant leaf tissue.

For example, the average dry weight to wet weight ratio for the20-week-old plants was 0.23, while the corresponding value for the4-week-old plants was 0.15. When this phenomenon is taken into accountand product accumulation is expressed on a fresh weight basis it becomesfar more apparent that pHBA levels did increase as the plants continuedto grow (FIG. 3B), except for the two CPL-expressing plants.

The 20-week-old primary transformants were large enough to screen forstalk levels of pHBA without damaging the plants. At this stage ofdevelopment, the oldest stem tissue is semi-mature and new tillersemerge. Since the stalk is the only part of the sugarcane plant that isnormally harvested in the existing sugar mill infrastructure, pHBAaccumulation in this tissue is the most important gauge for technicalsuccess. Leaf and stem samples were taken from 20-week-old plants, andtotal pHBA was determined by HPLC after methanol extraction and acidhydrolysis. The third internode from the bottom of the plant was thesource of stem tissue for this analysis, and the leaf samples wereobtained from the third fully unfurled leaf from the top of the plant.Generally speaking, leaf levels of pHBA were considerably higher thanstalk levels.

However, the difference was much more pronounced for the CPL-expressingplants. For example, the average stalk to leaf ratio of pHBA for thefive UH lines that were examined was 0.324±0.031, and the highest stalklevel of pHBA was 0.24% DW, which is equivalent to 0.52% pHBA glucoseconjugates. In marked contrast, the corresponding ratios for UC63 andUC65 were 0.135 and 0.133, respectively, and product accumulation in thestalk of the best plant (UC63) was only 0.06% DW. Since there are noreported values in the literature for pHBA levels in stem tissue fortransgenic plants expressing CPL or HCHL, it will be very interesting tosee if these observations will extend to other plant systems.Nevertheless, taken together the above results suggest that HCHL is abetter catalyst for pHBA production in sugarcane than CPL, andsubsequent studies focused on the UH series of plants.

To gain a better understanding of pHBA accumulation in different partsthe plant, leaf and stem segments were sampled from the primary shoot of20-week-old UH1. The first leaf at the top with a fully visible dewlapwas designated “leaf 1” and consecutive leaves down the stalk werenumbered in ascending order. The stem segments were numbered similarlywith “internode 1” corresponding to the internode immediately above thepoint of attachment of leaf 1. Note that the values shown refer to totalpHBA after acid hydrolysis. Except for the youngest leaf examined,product accumulation in leaves was relatively uniform along the lengthof the plant achieving a maximum value of ˜1.0% DW. Product accumulationalso varied along the length of the leaf, with the tip of the leafhaving about twice as much pHBA as the base of leaf A similar trend wasobserved in the stalk, but there was a much larger discrepancy betweenyoung stem tissue and old stem tissue. In agreement with the resultsdescribed above, pHBA levels in mature stem tissue were about 3-foldlower than mature leaf tissue. These results add additional support tothe notion that pHBA accumulation in HCHL-expressing sugarcane plantsincreases as a function of time.

Additional insight on pHBA distribution was obtained from dissectionexperiments. Three different compartments of the stalk were examined:rind, pith, and vascular bundles. The most pHBA was found in the rind(1% DW), while the pith and vascular bundles had 3- to 4-fold lowerlevels. Indeed, pHBA levels in the rind were very similar to valuesobtained from the leaf midrib and leaf lamina

Of all of the HCHL-expressing primary transformants monitored, UH98consistently had the highest levels of pHBA in both leaf and stemtissue. When this plant was 20 weeks old pHBA accumulation in leaftissue was 2.8% DW (leaf lamina, 3.35% DW; leaf midrib, 1.61% DW). Thecorresponding value for mature stem tissue was 0.67% DW (rind, 0.96% DW;pith, 0.65% DW). Despite these very high levels of pHBA glucoseconjugates, UH98 was morphologically indistinguishable from thenon-transformed control line TC1 (FIG. 5).

The present invention contemplates a method for producing pHBA in a C4grass, the method comprising expressing one or more genetic sequencesencoding one or more pHBA biosynthetic enzymes in cells of a C4 grasssuch that pHBA accumulates anywhere in the cell or extra-cellular matrixof the plant.

Accordingly, reference herein to hydroxycinnamoyl-CoA hydratase/lyase orchorismate pyruvate lyase includes all homologs thereof.

In a preferred embodiment, the present invention contemplates a methodfor generating a plant which produces pHBA or a precursor thereof, saidmethod comprising introducing into cells of said plant a geneticsequence comprising at least one of the following:—

-   -   (i) a nucleotide sequence encoding hydroxycinnamoyl-CoA        hydratase/lyase;    -   (ii) a nucleotide sequence encoding chorismate pyruvate lyase;    -   (iii) a nucleotide sequence comprising the ubiC gene from E.        coli, or a homolg thereof; and/or    -   (iv) a nucleotide sequence comprising the HCHL gene from        Pseudomonas fluorescens or homolg thereof;        and then regenerating a plant from said cells.

Preferably, the plant is a C4 grass and, in a particularly preferredembodiment, sugarcane.

In order that pHBA may be produced in cells of a C4 grass, suitablesequences such as those encoding one or more pHBA biosynthetic enzymesmust be introduced into and expressed in the cells. That is, the plantneeds to undergo genetic modification so that the metabolites and/ormetabolic and/or biosynthetic pathways can be harnessed for theproduction of the pHBA or a precursor thereof. This may conveniently beachieved through the use of genetic constructs, engineered to comprisenucleotide sequences required to effect pHBA production.

Clearly, the genetic sequences may be modified to insert any leader,tail or signal sequence to direct the enzyme to an appropriate locationin the cell. In a preferred embodiment, the pHBA biosynthetic enzymesare targetted to the plastid.

To effect expression of the nucleotide sequence of the presentinvention, it may conveniently be incorporated into a chimeric geneticconstruct comprising inter alia one or more of the following: a promotersequence, a 5′ non-coding region, a cis-regulatory region such as afunctional binding site for transcriptional regulatory protein ortranslational regulatory protein, an upstream activator sequence, anenhancer element, a silencer element, a TATA box motif, a CCAAT boxmotif, an upstream open reading frame, transcriptional start site,translational start site, and/or nucleotide sequence which encodes aleader sequence, and a 3′ non-translated region. Preferable the chimericgenetic construct is designed for transformation of plants ashereinafter described.

The term “5′ non-coding region” is used herein in its broadest contextto include all nucleotide sequences which are derived from the upstreamregion of an expressible gene, other than those sequences which encodeamino acid residues which comprise the polypeptide product of said gene,wherein 5′ non-coding region confers or activates or otherwisefacilitates, at least in part, expression of the gene.

The term “gene” is used in its broadest context to include both agenomic DNA region corresponding to the gene as well as a cDNA sequencecorresponding to exons or a recombinant molecule engineered to encode afunctional form of a product.

As used herein, the term “cis-acting sequence” or “cis-regulatoryregion” or similar term shall be taken to mean any sequence ofnucleotides which is derived from an expressible genetic sequencewherein the expression of the first genetic sequence is regulated, atleast in part, by said sequence of nucleotides. Those skilled in the artwill be aware that a cis-regulatory region may be capable of activating,silencing, enhancing, repressing or otherwise altering the level ofexpression and/or cell-type-specificity and/or developmental specificityof any structural gene sequence.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences of a classicalgenomic gene, including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or environmental stimuli, or in a tissue-specific orcell-type-specific manner. A promoter is usually, but not necessarily,positioned upstream or 5′, of a structural gene, the expression of whichit regulates. Furthermore, the regulatory elements comprising a promoterare usually positioned within 2 kilobase pairs (kb) of the start site oftranscription of the gene.

In the present context, the term “promoter” is also used to describe asynthetic or fusion molecule, or derivative which confers, activates orenhances expression of a structural gene or other nucleic acid molecule,in a plant cell. Preferred promoters according to the invention maycontain additional copies of one or more specific regulatory elements tofurther enhance expression in a cell, and/or to alter the timing ofexpression of a gene to which it is operably connected.

The term “operably connected” or “operably linked” in the presentcontext means placing a gene under the regulatory control of a promoter,which then controls the transcription and optionally translation of thegene. In the construction of heterologous promoter/structural genecombinations, it is generally preferred to position the genetic sequenceor promoter at a distance from the gene transcription start site that isapproximately the same as the distance between that genetic sequence orpromoter and the gene it controls in its natural setting, i.e. the genefrom which the genetic sequence or promoter is derived. As is known inthe art, some variation in this distance can be accommodated withoutloss of function. Similarly, the preferred positioning of a regulatorysequence element with respect to a heterologous gene to be placed underits control is defined by the positioning of the element in its naturalsetting, i.e. the genes from which it is derived.

Promoter sequences contemplated by the present invention may be nativeto the host plant to be transformed or may be derived from analternative source, where the region is functional in the host plant.Other sources include the Agrobacterium T-DNA genes, such as thepromoters for the biosynthesis of nopaline, octapine, mannopine, orother opine promoters; promoters from plants, such as the ubiquitinpromoter; tissue specific promoters (see, e.g. U.S. Pat. No. 5,459,252;International Patent Publication No. WO 91/13992); promoters fromviruses (including host specific viruses), or partially or whollysynthetic promoters. Numerous promoters that are functional in mono- anddicotyledonous plants are well known in the art (see, for example,Greve, J. Mol. Appl. Genet. 1: 499-511, 1983; Salomon et al., EMBO J. 3:141-146, 1984; Garfinkel et al., Cell 27: 143-153, 1983; Barker et al.,Plant Mol. Biol. 2: 235-350, 1983); including various promoters isolatedfrom plants (such as the Ubi promoter from the maize ubi-1 gene, e.g.U.S. Pat. No. 4,962,028) and viruses (such as the cauliflower mosaicvirus promoter, CaMV 35S).

In the context of the present invention, a particularly usefultissue-specific promoter is one which drives expression specifically inthe stems of sugarcane plants. Such a stem-specific promoter is, forexample, that described in International Patent Publication No. WO01/18211.

The promoter sequences may include regions which regulate transcription,where the regulation involves, for example, chemical or physicalrepression or induction (e.g. regulation based on metabolites, light, orother physicochemical factors; see, e.g. International PatentPublication No. WO 93/06710 disclosing a nematode responsive promoter)or regulation based on cell differentiation (such as associated withleaves, roots, seed, or the like in plants; see, e.g. U.S. Pat. No.5,459,252 disclosing a root-specific promoter). Thus, the promoterregion, or the regulatory portion of such region, is obtained from anappropriate gene that is so regulated. For example, the ribulose1,5-bisphosphate carboxylase gene is light-induced and may be used fortranscriptional initiation. Other genes are known which are induced bystress, temperature, wounding, pathogen effects, etc.

The chimeric genetic construct of the present invention may alsocomprise a 3′ non-translated sequence. A 3′ non-translated sequencerefers to that portion of a gene comprising a DNA segment that containsa polyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. The polyadenylation signalis characterized by effecting the addition of polyadenylic acid tractsto the 3′ end of the mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon

The 3′ non-translated regulatory DNA sequence preferably includes fromabout 50 to 1,000 nucleotide base pairs and may contain planttranscriptional and translational termination sequences in addition to apolyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. Examples of suitable 3′non-translated sequences are the 3′ transcribed non-translated regionscontaining a polyadenylation signal from the nopaline synthase (nos)gene of Agrobacterium tumefaciens (Bevan et al., Nucl. Acid. Res. 11:369, 1983) and the terminator for the T7 transcript from the octopinesynthase gene of Agrobacterium tumefaciens. Alternatively, suitable 3′non-translated sequences may be derived from plant genes such as the 3′end of the protease inhibitor I or II genes from potato or tomato, thesoybean storage protein genes and the pea E9 small sub-unit of theribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene, although other3′ elements known to those of skill in the art can also be employed.Alternatively, 3′ non-translated regulatory sequences can be obtained denovo as, for example, described by An (Methods of Enzymology 153: 292,1987), which is incorporated herein by reference.

A genetic construct can also be introduced into a vector, such as aplasmid. Plasmid vectors include additional DNA sequences that providefor easy selection, amplification, and transformation of the expressioncassette in prokaryotic and eukaryotic cells, e.g. pUC-derived vectors,pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, orpBS-derived vectors. Additional DNA sequences include origins ofreplication to provide for autonomous replication of the vector,selectable marker genes, preferably encoding, for example, antibiotic orherbicide resistance or green fluorescent protein or other visiblemarkers, unique multiple cloning sites providing for multiple sites toinsert DNA sequences or genes encoded in the chimeric genetic construct,and sequences that enhance transformation of prokaryotic and eukaryoticcells.

The vector preferably contains an element(s) that permits either stableintegration of the vector or a chimeric genetic construct containedtherein into the host cell genome, or autonomous replication of thevector in the cell independent of the genome of the cell. The vector, ora construct contained therein, may be integrated into the host cellgenome when introduced into a host cell. For integration, the vector mayrely on a foreign or endogenous DNA sequence present therein or anyother element of the vector for stable integration of the vector intothe genome by homologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector or a constructcontained therein to be integrated into the host cell genome at aprecise location in the chromosome. To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to1,500 base pairs, preferably 400 to 1,500 base pairs, and mostpreferably 800 to 1,500 base pairs, which are highly homologous with thecorresponding target sequence to enhance the probability of homologousrecombination. The integrational elements may be any sequence that ishomologous with the target sequence in the genome of the host cell.Furthermore, the integrational elements may be non-encoding or encodingnucleic acid sequences.

For cloning and sub-cloning purposes, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously in ahost cell such as a bacterial cell. Examples of bacterial origins ofreplication are the origins of replication of plasmids pBR322, pUC19,pACYC177, and pACYC184 permitting replication in E. coli, and pUB110,pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. The originof replication may be one having a mutation to make its functiontemperature-sensitive in a Bacillus cell (see, e.g. Ehrlich, Proc. Natl.Acad. Sci. USA 75: 1433, 1978).

To facilitate identification of transformed cells, the vector desirablycomprises a further genetic construct comprising a selectable orscreenable marker gene. The actual choice of a marker is not crucial aslong as it is functional (i.e. selective) in combination with the plantcells of choice. The marker gene and the nucleotide sequence of interestdo not have to be linked, since co-transformation of unlinked genes as,for example, described in U.S. Pat. No. 4,399,216 is also an efficientprocess in plant transformation.

Included within the terms selectable or screenable marker genes aregenes that encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers that encode a secretable antigen that can be identifiedby antibody interaction, or secretable enzymes that can be detected bytheir catalytic activity. Secretable proteins include, but are notrestricted to, proteins that are inserted or trapped in the cell wall(e.g. proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S); small, diffusible proteinsdetectable, for example, by ELISA; and small active enzymes detectablein extracellular solution such as, for example, α-amylase, β-lactamase,phosphinothricin acetyltransferase).

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, erythromycin, chloramphenicolor tetracycline resistance. Exemplary selectable markers for selectionof plant transformants include, but are not limited to, a hyg gene whichencodes hygromycin B resistance; a neomycin phosphotransferase (npt)gene conferring resistance to kanamycin, paromomycin, G418 and the likeas, for example, described by Potrykus et al. (Mol. Gene. Genet. 199:183, 1985); a glutathione-S-transferase gene from rat liver conferringresistance to glutathione derived herbicides as, for example, describedin EP-A 256 223; a glutamine synthetase gene conferring, uponoverexpression, resistance to glutamine synthetase inhibitors such asphosphinothricin as, for example, described International PatentPublication No. WO 87/05327, an acetyl transferase gene fromStreptomyces viridochromogenes conferring resistance to the selectiveagent phosphinothricin as, for example, described in European PatentApplication No. EP-A 275 957, a gene encoding a5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance toN-phosphonomethylglycine as, for example, described by Hinchee et al.(Biotech 6: 915, 1988), a bar gene conferring resistance againstbialaphos as, for example, described in International Patent PublicationNo. WO 91/02071; a nitrilase gene such as bxn from Klebsiella ozaenaewhich confers resistance to bromoxynil; a dihydrofolate reductase (DHFR)gene conferring resistance to methotrexate (Thillet et al., J. Biol.Chem. 263: 12500, 1988); a mutant acetolactate synthase gene (ALS),which confers resistance to imidazolinone, sulfonylurea or otherALS-inhibiting chemicals (European Patent Application No. EP-A-154 204)or a mutated anthranilate synthase gene that confers resistance to5-methyl tryptophan.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known; a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known; an aequorin gene(Prasher et al., Biochem. Biophys. Res. Comm. 126: 1259, 1985), whichmay be employed in calcium-sensitive bioluminescence detection; a greenfluorescent protein gene (Niedz et al., Plant Cell Reports 14: 403,1995); a luciferase (luc) gene (Ow et al., Science 234: 856, 1986),which allows for bioluminescence detection; a β-lactamase gene(Sutcliffe, Proc. Natl. Acad. Sci. USA 75: 3737, 1978), which encodes anenzyme for which various chromogenic substrates are known (e.g. PADAC, achromogenic cephalosporin); an R-locus gene, encoding a product thatregulates the production of anthocyanin pigments (red colour) in planttissues (Dellaporta et al., in Chromosome Structure and Function pp.263-282, 1988); an α-amylase gene (Ikuta et al., Biotech 8: 241, 1990);a tyrosinase gene (Katz et al, J. Gen. Microbiol. 129: 2703, 1983) whichencodes an enzyme capable of oxidizing tyrosine to dopa and dopaquinonewhich in turn condenses to form the easily detectable compound melanin;or a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA 80: 1101,1983), which encodes a catechol dioxygenase that can convert chromogeniccatechols.

A further aspect of the present invention provides a transfected ortransformed cell, tissue, or organ from a C4 grass, which comprises anucleotide sequence encoding one or more enzymes required for theproduction of a useful product.

The vectors and chimeric genetic construct(s) of the present inventionmay be introduced into a cell by various techniques known to thoseskilled in the art. The technique used may vary depending on the knownsuccessful techniques for that particular organism.

Techniques for introducing vectors, chimeric genetic constructs and thelike into cells include, but are not limited to, transformation usingCaCl₂ and variations thereof, direct DNA uptake into protoplasts,PEG-mediated uptake to protoplasts, microparticle bombardment,electroporation, microinjection of DNA, microparticle bombardment oftissue explants or cells, vacuum-infiltration of tissue with nucleicacid, and T-DNA-mediated transfer from Agrobacterium to the planttissue.

For microparticle bombardment of cells, a microparticle is propelledinto a cell to produce a transformed cell. Any suitable ballistic celltransformation methodology and apparatus can be used in performing thepresent invention. Exemplary apparatus and procedures are disclosed byStomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat.No. 4,945,050). When using ballistic transformation procedures, thegenetic construct may incorporate a plasmid capable of replicating inthe cell to be transformed.

Examples of microparticles suitable for use in such systems include 0.1to 10 μm and more particularly 0.5 to 5 μm tungsten particles or goldspheres. The DNA construct may be deposited on the microparticle by anysuitable technique, such as by precipitation.

Plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a chimericgenetic construct of the present invention and a whole plant generatedtherefrom. The particular tissue chosen will vary depending on theclonal propagation systems available for, and best suited to, theparticular species being transformed. Exemplary tissue targets includeleaf disks, embryos, cotyledons, hypocotyls, megagametophytes, callustissue, existing meristematic tissue (e.g. apical meristem, axillarybuds, and root meristems), and induced meristem tissue (e.g. cotyledonmeristem and hypocotyl meristem).

The regenerated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give a homozygous second generation (or T2) transformant, and the T2plants further propagated through classical breeding techniques.

Even more particularly, the present invention provides a plant cell ormulticellular plant or progeny thereof wherein said cell, plant, progenyor part thereof exhibits an activity to manufacture PHAs.

The term “genetically modified” is used in its broadest sense andincludes introducing gene(s) into cells, mutating gene(s) in cells andaltering or modulating the regulation of gene(s) in cells. In thecontext of the present invention, a transgenic cell or plant line mayalso be considered as a mutant cell or plant line when compared with itsnon-transgenic counterpart. In essence, a selected plant is firstgenetically modified to introduce a genetic sequence encoding a desiredproduct or intermediate.

Where genetic sequences for more than one gene are to be used in theperformance of the present invention, they may be introducedsimultaneously or sequentially, separately or together, into the targetcells that are to be transformed. For example, a singe genetic constructmay comprise all the required genetic sequences for the practice of thesubject invention, and this single construct may be introduced into thecells via any number of different means, as discussed below. Moreover,each genetic sequence may be operable linked to and under the control ofits own promoter, or may be comprised within a single polycistronicunit. Alternatively, separate genetic constructs may be utilized, eachcomprising one of the needed genetic sequences. In this event, more thanone construct may be introduced into the target cells simultaneously orsequentially. Here and elsewhere throughout the subject specification,the terms “target cells” and “cells to be transformed” should beregarded as being synonymous and refer to cells of a C4 grass that areto be used in accordance with the present invention as a bioreactor.

The one or more genetic sequences, introduced into a C4 grass plantcell, need to be expressed in order to enable the manufacture andaccumulation of a product. The term “expression” is to be construed inits broadest sense and includes and encompasses transcription andtranslation of a genetic sequence to a translation product.

Some plant cells may already comprise a homolog of one or more of thegenetic sequences encoding enzymes needed for the production of a givenproduct. In instances where a target plant cell, such as a sugarcanecell, already comprises one or more suitable genetic sequences, capableof directing sufficiently high expression, only those enzymes missing ina given pathway need be provided through via a genetic construct asdescribed above.

The present invention extends to homologs and derivatives of anysuitable sequences, whether found naturally in a target cell or providedexogenously having been derived from another plant, animal, protist,fungal, archeal or bacterial source, inter alia. The derivatives may beat the protein or nucleic acid level.

By “derivative” in relation to a polypeptide is meant a polypeptide thathas been derived from the basic sequence by modification, for example,by conjugation or complexing with other chemical moieties or bypost-translational modification techniques as would be understood in theart. The term “derivative” also includes within its scope alterationsthat have been made to a parent sequence including additions, ordeletions that provide for functionally-equivalent molecules.Accordingly, the term “derivative” encompasses molecules that affect aplant's phenotype in the same way as does the parent an amino acidsequence from which it was generated. Also encompassed are polypeptidesin which one or more amino acids have been replaced by different aminoacids. It is well understood in the art that some amino acids may bechanged to others with broadly similar properties without changing thenature of the activity of the polypeptide (conservative substitutions)as described hereinafter. These terms also encompass polypeptides inwhich one or more amino acids have been added or deleted, or replacedwith different amino acids.

“Polypeptide”, “peptide” and “an amino acid sequence” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues thereof. Thus, these terms apply toamino acid polymers in which one or more amino acid residues is asynthetic non-naturally-occurring amino acid, such as a chemicalanalogue of a corresponding naturally-occurring amino acid, as well asto naturally-occurring amino acid polymers.

The term “derivative” also encompasses fragments. A “fragment”, as usedherein, means a portion or a part of a full-length parent polypeptide,which retains the activity of the parent polypeptide. As used herein,the term “biologically-active fragment” includes deletion mutants andsmall peptides, for example, of at least 10, preferably at least 20 andmore preferably at least 30 contiguous amino acids, which comprise theabove activity. Peptides of this type may be obtained through theapplication of standard recombinant nucleic acid techniques orsynthesized using conventional liquid or solid phase synthesistechniques. For example, reference may be made to solution synthesis orsolid phase synthesis as described, for example, in Chapter 9 entitled“Peptide Synthesis” by Atherton and Shephard which is included in apublication entitled “Synthetic Vaccines” edited by Nicholson andpublished by Blackwell Scientific Publications. Alternatively, peptidescan be produced by digestion of an amino acid sequence of the inventionwith proteinases such as endoLys-C, endoArg-C, endoGlu-C andstaphylococcus V8-protease. The digested fragments can be purified by,for example, high performance liquid chromatographic (HPLC) techniques.Any such fragment, irrespective of its means of generation, is to beunderstood to be encompassed by the term “derivative” as used herein.

The terms “variant” and “homolog” refer to nucleotide sequencesdisplaying substantial sequence identity with a reference nucleotidesequences or polynucleotides that hybridize with a reference sequenceunder stringency conditions that are defined hereinafter. The terms“nucleotide sequence”, “polynucleotide” and “nucleic acid molecule” maybe used herein interchangeably and encompass polynucleotides in whichone or more nucleotides have been added or deleted, or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletionsand substitutions can be made to a reference nucleotide sequence wherebythe altered polynucleotide retains the biological function or activityof the reference polynucleotide. The term “variant” also includesnaturally-occurring allelic variants.

The extent of homology may be determined using sequence comparisonprograms such as GAP. In this way, sequences of a similar orsubstantially different length to those cited herein might be comparedby insertion of gaps into the alignment, such gaps being determined, forexample, by the comparison algorithm used by GAP, as is furtherdiscussed below.

Homologous sequences will generally hybridize under particular specifiedconditions. The term “hybridization” denotes the pairing ofcomplementary nucleotide sequences to produce a DNA-DNA hybrid or aDNA-RNA hybrid. Complementary base sequences are those sequences thatare related by the base-pairing rules. In DNA-DNA hybridization, A pairswith T and C pairs with G. In DNA-RNA hybridization, U pairs with A andC pairs with G. In this regard, the terms “match” and “mismatch” as usedherein refer to the hybridization potential of paired nucleotides incomplementary nucleic acid strands. Matched nucleotides hybridizeefficiently, such as the classical A-T and G-C base pair mentionedabove. Mismatches are other combinations of nucleotides that do nothybridize efficiently.

The extent of hybridization that may be displayed by homologoussequences depends on the conditions of, for example, temperature, ionicstrength, presence or absence of certain organic solvents, under whichhybridization and washing procedures are carried out. The higher thestringency, the higher will be the degree of complementarity betweenimmobilised target nucleotide sequences and the labelled probepolynucleotide sequences that remain hybridized to the target afterwashing. “High stringency conditions” refers to temperature and ionicconditions under which only nucleotide sequences having a high frequencyof complementary bases will hybridize. The stringency required isnucleotide-sequence dependent, and further depends upon the variouscomponents present during hybridization and subsequent washes, and thetime allowed for these processes. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m), is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T. In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences.

Reference herein to “low stringency conditions” is generally determinedat 42° C. and includes and encompasses from at least about 0% v/v to atleast about 15% v/v formamide, and from at least about 1 M to at leastabout 2 M salt for hybridization, and at least about 1 M to at leastabout 2 M for washing conditions. Alternative stringency conditions maybe applied where necessary, such as: medium stringency, which includesand encompasses from at least about 16% v/v to at least about 30% v/vformamide, and from at least about 0.5 M to at least about 0.9 M saltfor hybridization, and at least about 0.5 M to at least about 0.9 M saltfor washing conditions, or high stringency, which includes andencompasses from at least about 31% v/v to at least about 50% v/vformamide, and from at least about 0.01 M to least about 0.15 M salt forhybridization, and at least about 0.01 M to at least about 0.15 M saltfor washing conditions.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity” and “substantial identity”. A “reference sequence” isat least 12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e. only a portion ofthe complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e. gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e. resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., Nucl. AcidsRes. 25: 3389, 1997. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology” John Wiley & Sons Inc, 1994-1998, Chapter 15.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e. the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by theDNASIS computer program (Version 2.5 for windows; available from HitachiSoftware engineering Co., Ltd., South San Francisco, Calif., USA) usingstandard defaults as used in the reference manual accompanying thesoftware.

The one or more constructs may be introduced into a plant cell by anynumber of well-recognized means such as discussed above.

Preferably, the genetic constructs of the present invention areintroduced via the use of biolistics.

Accordingly, another aspect of the present invention provides atransgenic C4 grass, cells of which have been transformed with one ormore genetic sequences such that one of the following products in thecytosol, storage vacuole, non-plastid organelle or extra-cellular matrixof said cells:

-   -   (i) polyhydroxyalkanoates    -   (ii) vanillin    -   (iii) sorbitol    -   (iv) indigo    -   (v) fructans    -   (vi) lactic acid    -   (vii) adipic acid    -   (viii) 1,3-propanediol    -   (ix) 2-phenylethanol    -   (x) pHBA

The present invention extends to parts of plants tissue includingleaves, stems, vascular bundles, bark, reproductive material, roots andany extracted liquid (“juice”) from said plant.

While the present invention is exemplified using the compoundshereinbefore described, it is to be understood that the inventionextends to and encompasses the use of any suitable genetic sequencecapable of effecting the production of any product in the cells orextracellular matrix of a C4 grass.

The term “gene” is used in its broadest sense and includes cDNAcorresponding to the exons of a gene. Accordingly, reference herein to a“gene” is to be taken to include:—

-   (i) a classical genomic gene consisting of transcriptional and/or    translational regulatory sequences and/or a coding region and/or    non-translated sequences (i.e. introns, 5′- and 3′-untranslated    sequences); or-   (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons)    and 5′- and 3′-untranslated sequences of the gene.

The term “gene” is also used to describe synthetic or fusion moleculesencoding all or part of an expression product. In particularembodiments, the term “nucleic acid molecule” and “gene” may be usedinterchangeably.

In order to improve the efficiency and accumulation rate of a product, asuitable genetic sequence or sequences may be more specifically targetedso as to facilitate generation of expression products in particularsub-cellular areas or organelles within the plant. These include, forexample, the cytosol, a storage vacuole or a plastid or non-plastidorganelle.

The usefulness of a given sub-cellular compartment for a given productdepends on the nature and potential toxicity of the product to theplant. For example, PHA production is dependent on both the types ofpolymer produced and the metabolic pathways being engineered.

One particularly useful sub-cellular area for the production of productssuch as PHB, 1,3-propanediol and sorbitol, is the cytosol, whereinsucrose is both synthesized via gluconeogenesis and degraded viaglycolysis, leading to the production of pyruvate. In the cytosol, PHBis the preferred polymer, as moderate amounts of acetyl-CoA areavailable for phaA, phaB and phaC. A particularly useful sub-cellularorganelle is the mitochondrion, wherein acetyl-CoA, which may beproduced from pyruvate via mitochondrial pyruvate dehydrogenase, and/orperhaps from fatty acids via β-oxidation, is used to fuel the TCA cycle.A second useful sub-cellular organelle, with moderate to highacetyl-CoA, is the peroxisome, the site of fatty acid degradation viaβ-oxidation. These pathways involve the utilization of substantialamounts of acetyl-CoA, depleting reserves and rendering it unavailablefor use in manufacture of a product. However, the pyruvate needed foracetyl-CoA production is generated via glycolysis, which, in a sinktissue such as sugarcane stems, is fuelled by sucrose. Hence, the carbondrain that usually results from effecting the production of a product,such as a PHA, vanillin and the like, in a plant cell, is able to beovercome by the sucrose-accumulating plant cell's ability to mobiliseits substantial sucrose stores. The deleterious effects resulting fromproduct accumulation observed in non-sucrose-accumulating plant speciesdoes not occur in sugarcane, as a concomitant state of generalstarvation is precluded by the mobilization of sucrose from storagevacuoles, which replenishes the reduction of cellular acetyl-CoA poolscaused by the introduced genetic sequences.

Accumulation of PHAs, and other products (particularly products such aspHBA, adipic acid and indigo) may also be targeted to a plastid, such asa chloroplast, where large amounts of acetyl-CoA are used for fatty acidbiosynthesis. For example, for the production of PHAs other than PHB,plastids and peroxisomes are the preferred sub-cellular compartment, asPhaG (plastid) and PhaJ (peroxisome) provide monomers suitable forMCL-PHA polymerases such as PhaC1 from intermediates in fatty acidbiosynthesis and β-oxidation. In addition, particular biosyntheticpathways from which a particular product may be derived may exist onlyin the plastid. For example, the shikimate pathway is locatized in theplastid in plants. Products such as indigo and adipic acid may bederived from intermediates of the shikimate pathway via the addition ofnew biosynthetic enzymes. For these new enzymes to produce the productof interest, they must be localised to the particular organelles wheretheir substrates would be found.

In order to direct product accumulation to a desired sub-cellularlocation, particular specific “target sequences” may be incorporatedinto the genetic constructs described above.

A target sequence includes a signal sequence such as a signal sequenceto direct the protein to a plastid, vacuole, mitochondrion or otherappropriate organ or tissue.

Preferably, accumulation of PHA is in the cytosol or mitochondrion,assisted via mobilization of sucrose reserves located in the storagevacuoles of sugarcane stem cells.

Preferably, accumulation of adipic acid and indigo is in the plastid,wherein the introduced biosynthetic enzymes have access to intermediatesof the shikimate pathway.

The plants of the present invention may also be further “tagged” with areporter that identifies the plant as a plant bioreactor. Any number ofphysiological or genetic tags would be suitable, and readily identifiedby one of skill in the art. Examples of physiological “tags” that couldbe introduced include marker genes such as the green fluorescent proteingene, the firefly luciferase gene and the GUS gene.

Marker genes that alter the physical appearance of the plant may also beused as identifying tags. Examples include increased or decreased lengthof stems and/or alterations to color. Furthermore, a number ofresistance phenotypes may also be used to identify the plantbioreactors. Genes encoding resistance to pests such as bacterial,fungal or nematode pests have been identified in the art, and would besuitable as “tags”.

In addition, a genetic sequence itself may comprise the tag, referred toherein as “DNA barcoding”. Tagging in this manner is done by introducinga known non-coding polynucleotide sequence into the plant. The tag maythen be amplified from the plant using known PCR primers. Plants maythen be identified as bioreactors according to the present invention bythe presence of a particular size amplicon after the PCR reaction.Further discrimination, for example between types of plant bioreactor,may be achieved by altering the polynucleotide sequence of the DNAbarcode in the region between the PCR primers. In this way, the sequenceof the barcode may be elucidated using automated sequencing techniquesto determine the exact identity of the plant. This technique allows fora generic test to identify all plant bioreactors, and allows furtherdiscrimination to identify the type of bioreactor based on the sequenceof the DNA barcode.

The genetic sequences comprising or encoding the “tag” may be introducedto the plant using the methods hereinbefore described. The tag may beintroduced on the same construct as the biosynthetic gene, or may beindependently introduced. If introduced independently, the tag may beintroduced on a different construct at the same time as transformationwith the biosynthetic gene, or introduced to the plant before or afterthe biosynthetic gene.

Accordingly, the present invention contemplates a plant suitable for useas a bioreactor that has been tagged with a genetic sequence whichencodes or comprises a genotypic or phenotypic feature that allowsdifferentiation of the plant bioreactor from a wild-type plant.

Preferably the plant is a C4 grass, and more preferrably, the plant issugarcane.

The plant-based bioreactor system of the present invention is useful inenabling the production of molecules such as PHAs, pHBA, vanillin,sorbitol, indigo, fructans, lactic acid, adipic acid, 1,3-propanediol,2-phenylethanol, inter alia, by a number of different parties such asdifferent commercial entities. The present invention extends, therefore,to a data processing system to monitor the use of the plants and/or theproduction of target molecules.

Accordingly, another aspect of the present invention contemplates amethod for generating a target molecule in a sucrose-accumulating plant,said method comprising:—

-   (i) providing a plant or cells of a plant to a party; and-   (ii) permitting the party to generate and harvest molecules from    said plant or cells of said plant receiving and processing data from    said party.

The data received from the party includes, for example, numbers ofplants grown and/or harvested, the types of genetic constructsintroduced into the cells and/or income received from sale of theproducts.

The present invention is further described by the following non-limitingExamples.

Example 1 Materials

Restriction digests, DNA ligations and all other DNA manipulations wereperformed as described in Sambrook, et al., Molecular Cloning, ALaboratory Manual, 2n^(d) edition, Cold Spring Harbor Press, 1989.

Example 2 Cloning of the phaC1 Gene from P. aeruginosa

The phaC1 gene targeted to plant peroxisomes inserted into pART27 as anEcoRI/XbaI fragment, was obtained from Y. Poirier (University ofLausanne, Switzerland). In order to express the gene in sugarcane, itwas excised with the said enzymes, end-filled with T4 DNA polymerase(Promega) and inserted into the SmaI site of pUBI-MCS-Nos. To achievetargeting of the phaC1 gene product to mitochondria and plastids, thegene is modified as described below.

Example 3 Generation of Genetic Constructs (a) Constructs ComprisingSequences Encoding PHB-Synthesizing Enzymes

Constructs containing the phaA, phaB and phaC genes from Ralstoniaeutropha targeted to plastids and cloned in pUC18 as XbaI-SacI fragmentswere obtained from Y. Poirier (University of Lausanne, Switzerland).

The phaA, phaB and phaC genes derived from Ralstonia eutropha werecloned into the vector pU3z. This vector is a derivative of pGEM3(Promega) containing the maize polyubiquitin promoter and nos terminatorfrom A. tumefaciens, and works well as an expression vector insugarcane. All genes were amplified/modified using the polymerase chainreaction (PCR) prior to insertion into pU3z except phaC1, which wasblunt-end cloned into the same vector. All constructs used for planttransformation are listed below. Where inserts are modified, they aresequenced in full to ensure quality.

PCR modifications were performed as follows. Platinum Pfx (registeredtrademark) DNA polymerase, 10× buffer and PCR-enhancer were obtainedfrom Invitrogen. Final concentrations were one unit of polymerase perreaction, 1× buffer and enhancer, 2 mM Mg²⁺, 0.2 mM dNTP, 0.4 μM of eachprimer. All primers were purchased from Geneworks, Australia. Reactionswere performed in a MJC PTC-100 thermal cycler. The profile was initialdenaturation at 96° C. for 5 min, followed by 35 cycles of 94° C. for 30seconds, 42° C. for 30 sec and 72° C. for 2.5 min. a final extensionstep of 72° C. for 10 min preceded a final hold at 4° C. Table 1 listsprimers used in PCR reactions.

TABLE 1 POSITION, GENE, NAME SEQUENCE TARGET TphaFN₆ggatccatggcttctatgatatcct 5′, phaA-C, plastid [SEQ ID NO: 34] PhaFN₆GGATCCATGACTGACGTTGTCATC 5′, phaA, cytosol [SEQ ID NO: 35] PhbFN₆GGATCCATGACTCAGCGCATTGCG 5′, phaB, cytosol [SEQ ID NO: 36] PhcFN₆GGATCCATGGCGACCGGCAAAGGC 5′, phaC, cytosol [SEQ ID NO: 37] PhaRCTGAGTCATGTCCACTCC 3′, phaA, cytosol and [SEQ ID NO: 38] plastid PhbRCTGCCGACTGGTGGAACC 3′, phaB, cytosol and [SEQ ID NO: 39] plastid PhcRGAAGCGTCATGCCTTGGC 3′, phaC, cytosol and [SEQ ID NO: 40] plastid PhaC1CFN₆GGATCCATGAGCCAGAAGAAC 5′, phaC1, cytosol and [SEQ ID NO: 41]mitochondia PhaC1CR N₆GGTACCTCATCGTTCATGCACG 3′, phaC1, cytosol and[SEQ ID NO: 42] plastid PhaC1PF N₆CCCGGGTGAGCCAGAAGAACAATAAC5′, phaC1, plastid [SEQ ID NO: 43] PhaJF GGATCCATGAGCGCACAATCCCTGG5′, phaJ, peroxisome [SEQ ID NO: 44] PhaJR AAGCTTTTGAAGGCAGCTTGACCACGGC3′, phaJ, peroxisome [SEQ ID NO: 45] PhaGF CCCGGGTGAGGCCAGAAATCGCTGTAC5′, phaG, plastid [SEQ ID NO: 46] PhaGR GGTACCTCAGATGGCAAATGCATGC3′, phaG, plastid [SEQ ID NO: 47] SSP-FNNGAGCTCGATGGGAGGTGCTCGAAGACATATTA 5′, stem-specific CC promoter[SEQ ID NO: 48] SSP-R NNGGATCCTGTACTAGATATGGCAGC 3′, stem-specific[SEQ ID NO: 49] promoter

Approximately 10 ng of construct was used as template in each reaction.

Following PCR, fragments were gel purified and cloned into the BamHI andSmaI sites of pUSN in the correct orientation between the maizepolyubiquitin promoter and the nos-terminator from A. tumefaciens.Plasmid constructs were fully sequenced and purified by anion-exchangechromatography (Qiagen, Australia) prior to transformation into callustissue.

(b) Constructs Comprising Marker Sequences

Two plasmids were obtained from the CSIRO, Brisbane, Australia. Thefirst comprised the <pUbi-gfp-nos> construct, which carries the greenfluorescent protein (GFP) from Aquorea victoria under the control of thesame promoter as above. The second plasmid, designated “pEmuKn”,harbours an aphA gene (neomycin phosphotransferase) under the control ofthe Emu promoter. These plasmids were used without any furthermodification.

Example 4 Sugarcane Transformation (a) Generation of Embryogenic CallusTissue

Embryogenic callus of the sugarcane variety Q117 was established, asdescribed in Bower et al., Molec. Breeding 2: 239-249, 1996. Briefly,embryogenic callus was established by excision of inner leaf whorls fromcane tops 2-5 cm above the apical meristem. Disks of approximately 2 mmthickness were placed on MSC₃ medium that contains 3 μg/ml2,4-Dichlorophenoxy-acetic acid (2,4-D) and incubated at 28° C. in thedark for 2-5 months, with fortnightly subculturing. In order to avoidproblems associated with stress arising from the tissue culture process,such as somaclonal variation, unused callus tissue was discarded after 6months in culture.

Prior to transformation, embryogenic callus was transferred to osmoticMSC₃, as previously described (Bower et al., 1996, supra)

(b) Bombardment of Callus Tissue

DNA was coated onto tungsten particles (Sylvania M-10) and embryogeniccallus bombarded as described by Bower et al. (1996, supra).

Callus tissue was co-transformed with up to five individual constructs,plasmid solutions being mixed to give equimolar concentrations tofacilitate co-integration and expression of genes required for PHBproduction. There is a strong correlation between co-transformation andco-integration of constructs into the genome of plant hosts. Thecombinations of constructs introduced into Q117 callus and the targetfor products of each pha gene are shown in the following Table 2,wherein “cyt”=cytosol, “pla”=plastid, “mito”=mitochondrion and“perox”=peroxisome:

TABLE 2 COMBINATION AND TARGETING OF pha GENE PRODUCTS phaA (cyt), phaB(cyt), phaC (cyt), Ubi-GFP, Emu-Kn phaA (pla), phaB (pla), phaC (pla),Ubi-GFP, Emu-Kn phaA (cyt), phaB (cyt), phaC1 (cyt), Ubi-GFP, Emu-KnphaC1 (perox), Ub-GFP, Emu-Kn phaA (cyt), phaB (cyt), phaC (cyt), phaC1(perox), phaA (pla), phaB (pla), phaC (pla), phaG (pla), phaC1 (pla),Ubi-GFP, Emu-Kn phaC1 (perox), phaA (pla), phaB (pla), phaC (pla), phaG(pla), phaC1 (pla), Ubi-GFP, Emu-Kn phaA (pla), phaB (pla), phaC (pla),phaG (pla), phaC1 (pla), Ubi-GFP, Emu-Kn phaG (pla), phaC1 (pla),Ubi-GFP, Emu-Kn phaA (mito), phaB (mito), phaC (mito), Ubi-GFP, Emu-Kn

Microprojectile bombardment was performed as described in Bower et al.(supra), except that, to improve transformation efficiencies, the vacuumchamber was evacuated to −100 kPa atmospheric pressure and particlesaccelerated by a helium pulse of 3000 kPa for 100 ms.

(c) Selection of Transformed Material

Following bombardment, callus was allowed to recover for one hour,before being placed onto MSC₃ medium supplemented with 50 ng/mlGeneticin (registered trademark) (Invitrogen).

Putatively transformed tissue was screened for the expression of GFP andresistance to the antibiotic geneticin. Bombarded callus was examinedfor the presence of cells expressing GFP 7 days after transformation andstained in vivo with Nile Red, a sensitive in vivo stain specific forintracellular lipids, as previously described (Taguchi et al., FEMSMicrobiol. Lett. 198: 65-71, 2001; Greenspan, et al., 1985, supra). Forboth techniques, an Olympus SZX 12 stereomicroscope equipped with GFPexcitation and emission filters was used.

Dump

Antibiotic selection was continued for 3 months in the dark at 28° C.and continued during plant regeneration. Only calli expressing bothselectable markers were allowed to regenerate into plantlets.

(d) Regeneration of Transformed Sugarcane Plantlets

For plant regeneration, callus was transferred to medium without 2,4-Dand incubated at 24-26° C. under illumination. Plantlets appeared after2-4 months and were transferred to potting mix and kept inmini-glasshouse (Yates, Australia) for one week prior to transfer toglasshouse facilities.

Example 5 Determination of Quantity and Composition of PHA Produced

If the desired PHA is PHB, quantification in transgenic sugarcane isconducted by HPLC, as described by Karr et al., Applied andEnvironmental Microbiology 46: 1339-1344, 1983. This method allows forthe analysis of plant extracts with minimal handling. This technique isillustrated in Figure *.

For other PHAs, molecular characterization and quantification of PHAcontent in transgenic plants is carried out using gas chromatographyanalysis.

For GC analysis, PHA was separated from homogenized leaf samples bychloroform extraction, followed by methanol extraction, to remove lipidsother than PHA. The polymer was then purified further by acetoneextraction.

Transesterification of plant extracts was performed as described inBraunegg et al., Eur. J. Appl. Microbiol. Biotechnol. 6: 29-37, 1978modified by using boron trifluoride rather than sulfuric acid ascatalyst and decreased incubation time to 1 hr. Gas chromatographyanalysis was performed using a Varian 3300 chromatograph, as describedby Slater et al., in J. Bacteriol. 180: 667-73, 1998. Purified PHB(Coparsucar, Brazil) and methyl-3-hydroxybutyrate (Sigma) were used aspositive controls either pure or spiked into negative control plantextracts. Modifications to this method, namely using Boron trifluoriderather than sulfuric acid as catalyst and decreasing incubation times to1 hour, in addition to minor modifications to instrument parameters,were found to improve peak resolution.

Millenium software (Waters Corp., Milford, Mass.) is used to quantifyamounts of PHAs produced, by comparison of peak areas from plantextracts with those from standards of known concentration. GC-MS is usedto determine the composition of PHAs produced in transgenic sugarcane,as different hydroxy-alkanoates have different mass-spectrum signatures.

Example 6 Production of PHB in Transgenic Sugarcane Leaves

In plants accumulating high amounts of PHA, gene copy number,transcription levels and amount of protein are determined using standardmolecular biology techniques. Gene copy numbers are determined bySouthern blot analysis of genomic DNA from transformed plants producingPHAs. Transcription levels will be evaluated by northern blot analysisof RNA from transgenic plants and gene product levels examined bywestern blot analysis of protein extracts using antibodies against thegene products. The antisera were obtained from Prof. Y. Poirier(University of Lausanne, Switzerland).

Example 7 Targeting of PHB Production to Non Plastid SugarcaneOrganelles

The constructs pUbi-phaA, pUbi-phaB and pUbi-phaC comprising the phaA, Band C genes, respectively, transcriptionally fused to the aforementionedmaize polyubiquitin promoter were digested with BamHI anddephosphorylated with shrimp alkaline phosphatase (Promega, Maddison,USA). A BglII/BamHI fragment of the plasmid sB-pma-4-35S-β-del-GFP,containing the leader sequence and first 12 amino acid residues of the βsubunit of the Nicotiana plumbaginifolia mitochondrial F1-ATPase(Chaumont et al., PMB 24: 631-664, 1994), was ligated with pUbi-phaA, Bor C cut with BamHI. These constructs target PhaA, B or C to themitochondria with high efficiency. For targeting of pha gene products toother organelles, the genes were modified and ligated into plasmidsalready containing the required DNA targeting sequences.

Sugarcane was transformed with these constructs, using theaforementioned methods.

Transgenic plants thereby generated are screened for PHB production,using the aforementioned techniques.

Example 8 Production of PHB in Transgenic Sugarcane Stems

Plasmids pUbi-phaA, pUbi-phaB, pUbi-phaC, pUi-TP-phaA, pUbi-TP-phaB andpUbi-TP-phaC were digested with BamHI/EcoRI to release the pha geneswith or without the aforementioned plastid leader sequence at the 5′end, and with the aforementioned NOS terminator at the 3′ end. Thesefragments were ligated into the vector pSSP cut with BamHI/EcoRI. pSSPis a derivative of the vector p67G-420 (supplied by Prof R Birch,University of Queensland). p67G-420 houses, immediately upstream of aunique BamHI site, a stem-specific promoter isolated from sugarcane. Toobtain pSSP, a consensus ribosome binding site was removed from the 3′end of the promoter in the following way. The promoter was PCRamplified, using the aformentioned technique, with the primers SPP-F andSPP-R (see Table 1), incorporating BamHI and SacI sites into the 5′ and3′ ends of the promoter, respectively. The PCR product was digested withBamHI and SacI and then religated into the backbone of p67G_(—)420digested with the same enzymes. The promoter was fully sequenced toconfirm quality. The resulting constructs, which were fully sequenced toconfirm quality, drive gene expression in the stems and target the geneproducts to either the cytosol or the plastids. pSSP was linearized withBamHI and dephosphorylated with shrimp alkaline phosphatase, and ligatedwith the aforementioned BglII/BamHI fragment of the plasmidsB-pma-4-35S-β-del-GFP, giving the intermediate vector pSSP-Tm.BamHI/EcoRI fragments of pUbi-phaA, pUbi-phaB and pUbi-phaC were ligatedinto pSSP-Tm. The resulting constructs, which were fully sequenced toconfirm quality, drive gene expression in the stems and target the geneproducts to the mitochondria.

Sugarcane was transformed with these constructs using the aforementionedmethods.

Transgenic plants thereby generated are screened for PHB production,using the aforementioned techniques.

Example 9 Detection of PHB in Chloroplasts of Transgenic Sugarcane

Transgenic plants expressing PHB biosynthetic genes were producedaccording to the methods described herein. Accumulation of PHB in theplastid was assessed using both HPLC and transmission electronmicroscopy.

Figure * shows a graphical representation of the detection of PHB inchloroplasts of transgenic sugarcane. Panels A-C indicate detection ofPHB using HPLC. Panel A is a wild-type sugarcane control; Panel B is theplant in A spiked with PHB; Panel C depicts a transgenic sugarcane lineaccumulating PHB in the plastids. Arrows point to the elution point ofcrotonic acid, which is the product of acid-hydrolsis of PHB. The insertin Panel C shows that the peak at 30 min in C has the same spectrum ascrotonic acid.

Panels D-F in FIG. 4 show the detection of PHB granules in plants bytransmission electron microscopy. Panel D shows a positive controlcomprising a chloroplast from a mesophyll cell in a PHB +ve Arabidopsisplant (Bohmert et al. 2000). Panel E is a electron-micrograph showingPHB granules in a chloroplast of a mesophyll cell from a PHB-producingsugarcane plant. Panel F shows PHB granules in a chloroplast of abundle-sheath cell from the same plant line in E. Scale bars=200 nm.

Example 10 Agronomic Performance of PHB Producing Sugarcane Lines

Four transgenic sugarcane lines expressing the PHB biosynthesis genes ofRalstonia eutropha were grown for 3 months in a randomised glasshouseplot. Control plants comprised GFP-expressing andtissue-culture-regenerated wild-type plants. PHB content was assessed inlamina from the tips of mature leaves and quantified by HPLC analysis.

The results are shown in FIG. 5. The production of PHB in sugarcane atup to 1.6% of leaf dry-weight did not reduce agronomic performancecompared with GFP-expressing and wild-type control plants. Data are themean±SE (n=3). DW=dry-weight.

Example 11 Affect of PHB Production on Sugarcane Sugar Accumulation

The plants assessed in Example 10, ie. PHB producing, GFP expressing andwild-type sugarcane, were further examined for their sucrose, glucoseand fructose concentrations to determine the effect of PHB production onsugar content.

The results are shown in FIG. 6. It was observed that PHB accumulationof up to 1.6% of leaf dry-weight did not reduce sucrose, glucose,fructose or total sugar content in PHB producing (solid bars) plantscompared to GFP-expressing (open bars) and wild-type (hatched bars)controls. Data are the mean±SE (n=3). DW=dry-weight.

Example 12 Distribution of PHB in PHB Producing Sugarcane

The distribution of PHB throughout transgenic sugarcane line PHB3 wasdetermined by HPLC analysis. Samples were taken from:

-   -   (i) lamina of the tip, midpoint and base of young, intermediate        and mature leaves;    -   (ii) combined rind and pith of young, intermediate and mature        stem internodes; and    -   (iii) roots.

The PHB content data are presented as the mean percentage of leafdry-weight±SE (n=3). ND=not detected.

Example 13 Production of Vanillin in Sugarcane

Vanillin (4-hydroxy-3-methoxybenzaldehyde) would be produced as aco-product with sucrose. Sucrose yield is expected to decrease in directproportion to the amount of vanillin produced.

Initially, genes for the vanillin biosynthetic pathway from a knownsource are cloned. These genes are then expressed in sugarcane,including any tailoring of the expression pattern as required. Theproduct is produced as a glucose conjugate, which is stable.

A number of biological pathways have been discovered for thebiosynthesis/biodegradation of vanillin. At least 2 of these havesubstrates which are available in plants.

-   1) 3-Dehydroshikimic acid is produced as an intermediate in the    shikimate pathway. A pathway has been identified which converts this    substrate via 3-Dehydroshikimate dehydratase to protocatechuic acid    then to vanillic acid via Catechol-o-methyltransferase and finally    to vanillin via Aryl aldehyde dehydrogenase.-   2) Ferulic acid is a secondary metabolite of the phenylpropanoid    pathway involved in lignin synthesis. It is converted in planta to    feruloyl-CoA by feruloyl-CoA synthetase which in turn is converted    to vanillin by enoyl-CoA hydratase/aldolase.

Glucosylation of the product in vivo is expected to detoxify theproduct. Accordingly, inducible expression should not be required. Themaximum level of production is determined by the flux through thephenylpropanoid pathway. However, Sugarcane has a productivephenylpropanoid pathway and should adapt readily to increased demandsplaced on it for synthesis of vanillin.

Example 14 Production of Sorbitol in Sugarcane

Zymomonas mobilis is able to produce sorbitol from sucrose or a mixtureof glucose and fructose in a one-step reaction catalysed by theglucose-fructose oxidoreductase GFOR (Genbank accession no. Z80356,M97379). The glucose is oxidized to gluconolactone while the fructose isreduced to sorbitol.

glucose+fructose→sorbitol+gluconolactone

Sorbitol production in sugarcane could be achieved by using GFOR. Thisinvolves constructing an expression cassette by fusing GFOR to the maizepolyubiquitin promoter and nopaline synthase terminator and introducingthe cassette into sugarcane callus by biolistic transformaton. The Z.mobilis GFOR is not membrane-bound and resides in the periplasm andshould work equally well as a cytosolic enzyme in sugarcane.

Sorbitol production is unlikely to be toxic in sugarcane since sorbitolis found in numerous fruits (apples, pears, plums, berries, cherries).Sorbitol functions physiologically to regulate osmotic stress henceextremely high levels may be detrimental. Vacuolar storage maycircumvent this problem.

The threshold level at which sorbitol is deleterious to the host may bedetermined by growing sugarcane callus on solid medium containingsorbitol.

A potential large-scale system for the recovery of sorbitol fromsugarcane involves adding an aqueous organic salt solution, mixing andthen separating a salt water phase from a polyol-rich phase (seeinternational patent application WO210252).

Example 15 Indigo Production in Sugarcane

The chief incentive to use sugarcane as an indigo biofactory is toprovide a manufacturing route that will produce relatively inexpensiveindigo from a renewable feedstock.

Indigo production by microbial fermentation has been demonstrated byexpressing the genes that mediate indigo formation in E. coli (Drewlo2001, Berry 2002). The pigment is derived by converting endogenoustryptophan to indole using the Enterobacter aerogenes tryptophanase orL-tryptophan indole lyase EC 4.1.99.1 (Genbank accession no. D14297).Subsequently the indole is converted to indigo via two possiblereactions.

Route A: Pseudomonas putida napthalene dioxygenase (Genbank accessionno. M83949)

Route B: Ralstonia eutropha bec gene (Genbank accession no. AF306552)

Indigo production in sugarcane would involve constructing an expressioncassette by fusing the aforementioned genes to the maize polyubiquitinpromoter and nopaline synthase terminator and introducing the cassetteinto sugarcane callus by biolistic transformaton. Both route A and Bshould be tested if possible. Tryptophan is a product of the plantshikimate pathway, which is responsible for synthesizing ligninprecursors. The cloned genes will need to be plastid-targeted since theshikimate pathway reactions reside in this compartment. The availablemetabolic flux in this pathway is expected to be significant.

Example 16 Production of Fructans in Sugarcane

Naturally occurring fructans may contain 10 to 100,000 fructoseresidues. Bacteria produce the larger fructans whilst those occurring inplants are smaller. The larger polymers are desirable because they areless soluble in water and consequently easier to extract. Largerfructans will not affect the osmotic pressure in the cell to the samedegree as smaller molecules. Therefore it is possible to store greaterquantities of fructan before the cell is affected.

Numerous bacterial fructosyltransferases or levansucrases have beencharacterized (Genbank accession no. AY150365, Bacillus subtilis). Theseenzymes catalyze the transfer of the D-fructosyl residue from sucrose tothe β-2,6-linked residues of fructan.

Sucrose→fructan+glucose

Fructan production in sugarcane would be achieved by constructing anexpression cassette containing levansucrase, the maize polyubiquitinpromoter and nopaline synthase terminator and introducing the cassetteinto sugarcane callus by biolistic transformaton.

Levansucrase will probably require apoplastic or vacuolar targeting tomaximize access to substrate for conversion.

Fructan may then be recovered from sugarcane juice by ethanolprecipitation followed by vacuum-drying.

Example 17 Lactic Acid Production in Sugarcane

The production of Lactic acid (2-Hydroxypropanoic acid) in sugarcaneproceeds with the following steps:

-   -   (i) Obtain or clone lactate dehydrogenase (LDH) from a number of        sources, such a Lactobacillus spp. bacterium.    -   (ii) Expression of the gene in sugarcane, with any necessary        changes to the sequence such as codon preference. It is        preferred that the introduced gene is expressed in the cytosol,        therefore no targeting is required.    -   (iii) Regenerate plants and evaluate for lactate (or derivative)        production.

Lactic acid build-up may cause deleterious effect on cells. There areseveral ways by which cells can deal with this. One is to remove theacid either by diffusion or transport. The other is modification of theoffending chemical and export into the vacuole. Glycosylation is a majorsignal for this process and lactic acid possesses two potentialglycosylation sites.

Traditionally, lactic acid purification has been a complex chemicalprocess. However, recent advances have simplified this process and madeit significantly cheaper. It is anticipated that lactic acid can beremoved from the post-crushing millstream without great difficulty orextensive modification of existing structures. It is anticipated thatthe extraction process will be product dependent.

Example 18 Adipic Acid Production in Sugarcane

Adipic acid may be produced in sugarcane by one of two approaches.

I. Synthesis from Cis, Cis-Muconic Acid

Adipic acid has been produced in transgenic E. coli using the metabolicpathway illustrated below. Three genes were introduced into E. coli toproduce cis, cis-muconic acid that was subsequently purified from thefermentation broth and converted to adipic acid by catalytichydrogenation (step g, 10% Pt/C, H₂, 3400 kPa, 25° C.). This final stephas a 97% conversion efficiency.

The synthesis of cis, cis-muconic acid in sugarcane involves making useof the shikimate pathway. In order to use the shikimate pathway toproduce cis, cis-muconic acid the following biosynthetic enzymes, orhomologs thereof are introduced into sugarcane:

Klebsiella pneumoniae 3-dehydroshikimate dehydratase (aroZ)-enzyme d

3-dehydroshikimate protocatechuate

Klebsiella pneumoniae protocatechuate decarboxylase (aroY)-enzyme e

Protocatechuate→catechol

Acinetobacter calcoaceticus catechol 1,2-dioxygenase (catA)-enzyme f

Catechol+O₂→cis, cis-muconic acid

Introduction of these genes into sugarcane involves constructing anexpression cassette by fusing the genes described above to the maizepolyubiquitin promoter and nopaline synthase terminator and introducingthe cassette into sugarcane callus by biolistic transformation. Catecholis probably produced in most plants, and therefore, it may beunnecessary to clone additional copies of 3-dehydroshikimate dehydrataseor protocatechuate decarboxylase. The cloned gene(s) areplastid-targeted since the shikimate pathway reactions reside in thiscompartment.

The shikimate pathway executes a central role in plant secondarymetabolism. This is one of the most active pathways in plants in termsof carbon flux owing to the fact that it is the source of ligninprecursors. This makes it an attractive candidate for metabolicengineering.

II. Synthesis from Petroselinic Acid

Bio-based adipic acid can be obtained through ozonolysis (O₃) ofpetroselinic acid (18:1 Δ^(6 cis)), as shown in FIG. 10. The coproductlauric acid is also a potential source of feedstock for detergentmanufacture.

The seed oil of the coriander spice plant contains 80-90% petroselinicacid. A 36 kDa putative acyl-ACP desaturase (Genbank accession no.M93115) has been identified from coriander seed extracts and thecorresponding cDNA was able to confer the ability to producepetroselinic acid in tobacco callus (Cahoon 1992). Petroselinic acid wasquantified from extracted calli by gas chromatography and GC-MS (todetermine double bond position). Tobacco does not normally producepetroselinic acid hence the successful expression of the cDNA in tobaccosuggested that this desaturase was sufficient for petroselinic acidformation. This also infers that it may be feasible in sugarcane.

The metabolic pathway for producing petroselinic acid is unclear,however, evidence suggests that it is formed by the desaturation ofpalmitoyl-ACP by the 36 kDa desaturase followed by elongation to formpetroselinic acid (Cahoon 1994).

16:0-ACP→16:1Δ⁴-ACP→18:1Δ⁶-ACP

Recent studies have identified a 3-ketoacyl-ACP synthase (Genbankaccession no. AF263992) associated with the two-carbon elongation of16:1 Δ⁴-ACP.

Cis, cis-muconic acid in sugarcane juice would be converted to adipicacid by catalytic hydrogenation. The adipic acid in the resultantsolution can be recovered by solvent extraction. The solution iscontacted with chloroform or methylene chloride and the adipic acidrecovered in the aqueous fraction. The aqueous fraction would then beevaporated to yield crystalline adipic acid.

Example 19 Production of 1,3 propanediol (1,3-PD) in Sugarcane

1,3-PD is a natural product of glycerol fermentation in a fewenterobacteria and clostridia. Fermentation-derived 1,3-PD was notcommercially viable for many years due to the high cost of the glycerolfeedstock.

The metabolic reactions that convert glycerol to 1,3-PD have beenestablished from Klebsiella pneumoniae.

Klebsiella pneumoniae glycerol dehydratase (dhaB)

glycerol→3-hydroxypropionaldehyde+H₂O

Klebsiella pneumoniae 1,3-propanediol oxidoreductase (dhaT)

3-hydroxypropionaldehyde+NADH→1,3-propanediol+NAD

Sugarcane does not naturally produce glycerol therefore the reactionsthat convert triose phosphates to glycerol must also be engineered intosugarcane.

Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase

dihydroxyacetone phosphate+NADH→glycerol-3-phosphate+NAD

Saccharomyces cerevisiae glycerol-3-phosphatase

glycerol-3-phosphate+ADP→glycerol+ATP

Effectively, all four new genes must be cloned into sugarcane to convertit into a 1,3-PD biofactory. These genes will be assembled into anexpression cassette containing the maize polyubiquitin promoter andnopaline synthase terminator. The cassette will be introduced intosugarcane callus by biolistic transformation and expression will betargeted to the cytosol. The accumulation of 1,3-PD in plant tissue willbe assayed from plant extracts by conventional HPLC.

1,3-PD can be recovered from sugarcane juice by extraction withcyclohexane followed by vaporization of the residual solvent.Alternatively, distillation may be employed. Use of cyclohexane isenvironmentally unsound and distillation is energy intensive.Consequently, a method has been patented that describes the use of ionexclusion resins to recover 1,3-PD (see international patent applicationWO 01/73097).

Example 20 Production of 2-phenylethanol (2-PE) in Sugarcane

The production of 2-PE in sugarcane would be achieved in a similar wayto previous examples. Briefly, cloned genes for the 2-PE biosyntheticpathway, which has previously been determined, would be obtained.Second, these genes would then be expressed in sugarcane, tailoring theexpression pattern and codon usage if required. Finally, a stableproduct, as a glucose conjugate, is expected.

A biological pathway for the biosynthesis of 2-PE is presented in FIG.11.

The product is produced naturally in roses and hence should not betoxic. Glucosylation of the active group is likely to occur in sugarcaneto reduce potential toxicity.

2-PE would be recovered by crushing the cane and refining from juice asfor sucrose, and standard production processes for the synthetic formare well established.

2-PE is water-soluble and should be stable for the time normally takento process sugarcane for sucrose. If sugarcane stores 2-PE as a glucoseconjugate then alkaline hydrolysis may be required.

Example 21 Characterization of CPL-Expressing and HCHL-ExpressingSugarcane Plants

A chloroplast-targeted version of E. CPL situated between the maizeubi-1 promoter and nos terminator of the expression constructpU3z-mcs-nos, was co-bombarded with a plasmid containing a selectablemarker (pUKN) into embryogenic sugarcane callus to yield the UC seriesof transgenic lines. The UH series of plants was generated in the samemanner using an analogous expression construct that contained the ORF ofthe P. fluorescens HCHL gene. To serve as controls for the experimentsdescribed below, four non-transgenic lines (TC1-TC4) were alsoregenerated from the same callus material omitting the transformationand selection steps. The regenerated plants were grown in a greenhousefor four weeks and were then analyzed for pHBA accumulation in leaftissue using HPLC. Only plants that had higher levels than the controlplants (46% and 48% of the population for the UC lines and UH lines,respectively) were included in the analysis shown in FIG. 2.

Not surprisingly, none of the transgenic plants had significantly higherlevels of “free” pHBA than the control plants. Similar to the situationreported for tobacco plants expressing CPL (Siebert et al., PlantPhysiol. 112: 811-819, 1996) or HCHL (Mayer et al., Plant Cell13:1669-1682, 2001), the only two compounds that accumulated were pHBAglucose conjugates, a phenolic glucoside and a glucose ester. Bothcompounds contained a single glucose molecule that was attached by a1-O—-D linkage to the hydroxyl or carboxyl group of pHBA. Thepredominant product in all of the plants examined was the phenolicglucoside, which accounted for at least 90% of the pHBA (see below). Themean value for the population was 0.41%±0.04% of dry weight (DW), whichis almost 30-fold higher than the mean value for the non-transgeniccontrol plants 0.014%±0.01% DW. More important, the pHBA glucosidecontent of the best plant was 1.5% DW, which is equivalent to 0.69% DWfree pHBA after correcting for the attached glucose molecule. This valueis three times higher than the highest value obtained with transgenictobacco plants expressing a different chloroplast-targeted version ofCPL (Siebert et al., supra). The HCHL-expressing sugarcane plantsaccumulated even higher levels of pHBA. The mean value for total pHBAglucose conjugates in the UH lines was 0.70%±0.07% DW, and the highestlevel observed at this stage of development was 2.6% DW, which is verysimilar to the best value reported for transgenic tobacco plantsexpressing the same enzyme (Mayer et al., supra).

Based on the results obtained with the 4-week-old plants, a subset ofthe primary transformants was selected for further evaluation, and leaflevels of pHBA were determined after 16 weeks additional growth (FIG.15). Included in this analysis were the two CPL-expressing plants thatpreviously exhibited the highest levels of product accumulation (UC63and UC65) and five HCHL-expressing plants. The methanol-extractedsamples were subjected to acid hydrolysis, which quantitativelyhydrolyzes both pHBA glucose conjugates, and free pHBA was determined byHPLC.

It was anticipated that pHBA production would continue throughoutdevelopment and that the 20-week-old plants would have higher levels ofpHBA glucosides than the 4-week-old plants. However, the increase inpHBA content with age was not very dramatic nor was it universallyobserved when product accumulation was expressed on a dry weight basis.Part of the explanation for this is the lower water content of the olderplant leaf tissue. For example, the average dry weight to wet weightratio for the 20-week-old plants was 0.23, while the corresponding valuefor the 4-week-old plants was 0.15. When this phenomenon is taken intoaccount and product accumulation is expressed on a fresh weight basis itbecomes far more apparent that pHBA levels did increase as the plantscontinued to grow, except for the two CPL-expressing plants.

The 20-week-old primary transformants were large enough to screen forstalk levels of pHBA without damaging the plants. At this stage ofdevelopment, the oldest stem tissue is semi-mature and new tillersemerge. Since the stalk is the only part of the sugarcane plant that isnormally harvested in the existing sugar mill infrastructure, pHBAaccumulation in this tissue is the most important gauge for technicalsuccess. Leaf and stem samples were taken from 20-week-old plants, andtotal pHBA was determined by HPLC after methanol extraction and acidhydrolysis. The third internode from the bottom of the plant was thesource of stem tissue for this analysis, and the leaf samples wereobtained from the third fully unfurled leaf from the top of the plant.Generally speaking, leaf levels of pHBA were considerably higher thanstalk levels.

However, the difference was much more pronounced for the CPL-expressingplants. For example, the average stalk to leaf ratio of pHBA for thefive UH lines that were examined was 0.324±0.031, and the highest stalklevel of pHBA was 0.24% DW, which is equivalent to 0.52% pHBA glucoseconjugates. In marked contrast, the corresponding ratios for UC63 andUC65 were 0.135 and 0.133, respectively, and product accumulation in thestalk of the best plant (UC63) was only 0.06% DW. Since there are noreported values in the literature for pHBA levels in stem tissue fortransgenic plants expressing CPL or HCHL, it will be very interesting tosee if these observations will extend to other plant systems.Nevertheless, taken together the above results suggest that HCHL is abetter catalyst for pHBA production in sugarcane than CPL, andsubsequent studies focused on the UH series of plants.

Example 22 Localization of pHBA in Sugarcane Tissue

To gain a better understanding of pHBA accumulation in different partsthe plant, leaf and stem segments were sampled from the primary shoot of20-week-old UH1. The first leaf at the top with a fully visible dewlapwas designated “leaf 1” and consecutive leaves down the stalk werenumbered in ascending order. The stem segments were numbered similarlywith “internode 1” corresponding to the internode immediately above thepoint of attachment of leaf 1. The results from this analysis aresummarized in FIG. 15. Note that the values shown refer to total pHBAafter acid hydrolysis. Except for the youngest leaf examined, productaccumulation in leaves was relatively uniform along the length of theplant achieving a maximum value of ˜1.0% DW. Product accumulation alsovaried along the length of the leaf, with the tip of the leaf havingabout twice as much pHBA as the base of leaf (data not shown). A similartrend was observed in the stalk, but there was a much larger discrepancybetween young stem tissue and old stem tissue. In agreement with theresults described above, pHBA levels in mature stem tissue were about3-fold lower than mature leaf tissue. These results add additionalsupport to the notion that pHBA accumulation in HCHL-expressingsugarcane plants increases as a function of time.

Additional insight on pHBA distribution was obtained from dissectionexperiments similar to the one shown in FIG. 15. The plant that was usedfor this analysis was 20-week-old UH1. Three different compartments ofthe stalk were examined: rind, pith, and vascular bundles. The most pHBAwas found in the rind (1% DW), while the pith and vascular bundles had3- to 4-fold lower levels. Indeed, pHBA levels in the rind were verysimilar to values obtained from the leaf midrib and leaf lamina.

Of all of the HCHL-expressing primary transformants monitored, UH98(FIG. 2B) consistently had the highest levels of pHBA in both leaf andstem tissue. When this plant was 20 weeks old pHBA accumulation in leaftissue was 2.8% DW (leaf lamina, 3.35% DW; leaf midrib, 1.61% DW). Thecorresponding value for mature stem tissue was 0.67% DW (rind, 0.96% DW;pith, 0.65% DW). Despite these very high levels of pHBA glucoseconjugates, UH98 was morphologically indistinguishable from thenon-transformed control line TC1 (FIG. 5).

Example 23 Construction of cTP-CPL

PCR was used to generate the monocot chloroplast-targeting sequence thatwas fused to the N-terminus of E. coli CPL. The target for amplificationwas the maize rbcS gene (GenBank accession number Y00322), which codesfor the Rubisco small subunit precursor. Primer 1 (5′-CTA CTC ATA ACCATG GCG CCC ACC GTG-3′) hybridized to nucleotides 489-505 and introduceda NcoI site at the start codon of the transit peptide. Primer 2 (5′-CATCTT ACT CAT ATG CCG CAC CTG CAT GCA CCG GAT CCT TCC G-3′) hybridized tonucleotides 616-639 and introduced an NdeI site five amino acid residuesdownstream from the chloroplast cleavage site. The PCR product was cutwith NcoI and NdeI and inserted into pET24a-tTP-CPL (manuscript inpreparation), after the latter was cleaved with the same enzymes.pET24a-tTP-CPL contains the gene for a chimeric protein that consists ofthe tomato Rubisco small subunit transit peptide plus the first fouramino acid residues of the ‘mature’ Rubisco small subunit, fused to theN-terminus of E. coli CPL. The plasmid DNA was cut with NcoI and NdeI toremove the tomato chloroplast-targeting sequence, and this was replacedwith PCR-generated maize chloroplast-targeting sequence. The ligationmixture was introduced into E. coli DH10B, and growth was selected on LBmedia containing kanamycin (50 μg mL⁻¹). A representative plasmid

(pET24a-cTP-CPL) was sequenced and no PCR errors were found. Thepredicted chloroplast cleavage product of the cTP-CPL fusion protein isa CPL variant with five extra N-terminal amino acid residues (i.e.MQVRH-CPL).

Example 24 Generation of CPL and HCHL Expression Constructs Used forSugarcane Transformation

The antibiotic selection plasmid pUKN contains the ubi-1 promoter, theneomycin phosphotransferase gene and the nos terminator. The plasmidpU3z-mcs-nos was used for cTP-CPL and HCHL expression in sugarcane. Thisplasmid is a modification of pAHC20 and contains the maize ubi-1promoter and nos terminator. Both genes were inserted in the SpeI andKpnI sites of the multicloning region that immediately follows the maizeubi-1 intron. The gene coding for cTP-CPL was amplified frompET24a-cTP-CPL using primers 3 and 4. Primer 3 (5′-CTA CTC ATT TAC TAGTCA CCA TGG CGC CCA CCG TGA TG-3′) (SEQ ID NO: 50) hybridized to thefirst 18 nucleotides of the ORF of cTP-CPL and introduced a SpeI siteupstream from the start codon. Primer 3 also contained a consensusmonocot ribosomal binding site, CACC, which is situated between the SpeIsite and the initiator Met codon. Primer 4 (5′-CAT CTT ACT GGT ACC TTTAGT ACA ACG GTG ACG CC-3′) (SEQ ID NO: 51) hybridized to the other endof the insert and introduced a KpnI site just after the cTP-CPL stopcodon. The PCR product was cut with SpeI and KpnI, and ligated intosimilarly digested pU3z-mcs-nos. The ligation reaction mixture was usedto transform E. coli DH10B and growth was selected on LB mediacontaining ampicillin (100 μg mL⁻¹). A representative plasmid(pU3z-mcs-nos-cTP-CPL) was sequenced to confirm the absence of PCRerrors.

Primers 5 and 6 were used to amplify the Pseudomonas fluorescens strainAN103 HCHL gene (GenBank accession number Y13067) from plasmid pFI1039.Primer 5 (5′-CTA CTC ATT TAC TAG TCA CCA TGA GCA CAT ACG AAG GTC G-3′)(SEQ ID NO: 52) hybridized to the first 20 nucleotides of the ORF forHCHL and introduced a unique SpeI site upstream from the start codon.Primer 5 also contained a consensus monocot ribosomal binding site(CACC) that is situated between the SpeI site and the initiator Metcodon. Primer 6 (5′-CAT CTT ACT GGT ACC TTC AGC GTT TAT ACG CTT GCA-3′)(SEQ ID NO: 53) hybridized to the other end of the insert and introduceda KpnI site just after the HCHL stop codon. The PCR product was cut withSpeI and KpnI, and ligated into similarly digested pU3z-mcs-nos. Theligation reaction mixture was used to transform E. coli DH10B and growthwas selected on LB media containing ampicillin (100 μg mL⁻¹). Arepresentative plasmid (pU3z-mcs-nos-HCHL) was sequenced to confirm theabsence of PCR errors.

Example 25 Plant Transformation

Embryogenic callus from sugarcane cultivar Q117 was prepared essentiallyas described, and grown in the dark at 27° C. on MS media supplementedwith 3 mg L⁻¹ of 2,4-dichlorophenoxy acetic acid (2,4-D). The calli wereco-transformed with the antibiotic selection plasmid pUKN bymicroprojectile bombardment Following bombardment and a recovery phaseof 2 weeks in the dark, transformants were placed on MS-2,4D selectionmedia supplemented with 60 mg L⁻¹ geneticin. Individual callus clumpswere maintained separately throughout the selection process. After 6weeks, antibiotic-resistant calli were transferred to MS mediasupplemented with geneticin and incubated in the light to initiateplantlet regeneration. At least four plantlets per callus clump weretransferred to pots in a glasshouse certified for the physicalcontainment of transgenic plants for further analysis.

Example 26 Measurement of Accumulated Soluble Phenolics by HPLC

Soluble phenolics were extracted from 100-200 mg of leaf or stem tissue.The tissue samples was resuspended in 1 mL of 50% v/v methanol andhomogenized in a bead beater (Bio101/Savant, Fastprep FP120, Holbrook,N.Y.). The sample were then agitated in an orbital shaker (200 rpm) for1 hour at 37° C., and clarified by centrifugation. A 550-μL aliquot ofextract was transferred to a fresh tube and dried under vacuum, and thedry residue was dissolved in. 200 μL.

When the goal was to convert pHBA and vanillic acid glucose conjugatesto free pHBA and vanillic acid an acid hydrolysis step was included. A200-μL aliquot of the extract was transferred to a fresh tube and driedunder vacuum. After adding 200 μL of 1 N HCl to the dry residue andvortexing, the sample was incubated for 2 hours at 95° C. The sample wasthen neutralized by adding 200 μL of 1.2 N NaOH.

Soluble phenolics were detected by HPLC at 32° C. using the Novapak C18column described above. Samples were filtered through 0.2 μm syringefilters and 20 μL of filtrate was injected for each analysis. Mobilephases were the same as previously described. Solvent was pumped at 1 mLmin⁻¹ using the following gradient conditions: 0 min, 0% B; 80 min, 80%B; 81 min, 100% B; 85 min, 100% B; 86 min, 0% B. Total run time was 90minutes. An optimized gradient was applied to separate p-hydroxybenzoicacid and vanillic acid (0 min, 10% B; 20 min, 50% B; 21 min, 100% B; 24min, 100% B; 25 min, 10% B; total runtime was 35 minutes). Identifiedpeaks were quantified using authentic standards (Sigma-Aldrich Co.).

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto, or indicated in this specification, individually or collectively,and any and all combinations of any two or more of said steps orfeatures.

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1-54. (canceled)
 55. A method of producing a bioproduct in a geneticallymodified C4 grass which C4 grass substantially does not produce thebioproduct prior to genetic modification, said method comprisingintroducing a genetic sequence encoding an enzyme required for synthesisof the bioproduct or a precursor of said bioproduct into a cell or groupof cells of said C4 grass, regenerating a C4 grass from said cell orgroup of cells and growing the C4 grass or genetically modified progenytherefrom under conditions sufficient to produce the bioproduct.
 56. Themethod of claim 55 wherein the bioproduct is selected from the listcomprising a polyhydroxyalkanoate (PHA), p-hydroxybenzoic acid (pHBA),vanillin, sorbitol and fructan.
 57. The method of claim 56 wherein thebiproduct is polyhydroxyalkanoate (PHA) and the genetic sequencecomprises one or more genetic sequences selected from the listcomprising: (i) a nucleotide sequence encoding a phaA; (ii) a nucleotidesequence encoding phaB; (iii) a nucleotide sequence encoding phaC; (iv)a nucleotide sequence encoding phaC1; (v) a nucleotide sequence encodingphaG; (vi) a nucleotide sequence encoding phaJ; (vii) SEQ ID NO:1 or SEQID NO:3 or SEQ ID NO:10 or SEQ ID NO:12 or a nucleotide sequence havingat least 60% identity thereto after optimal alignment, or capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ IDNO:12 or a complementary form thereof under low stringency conditions;(viii) SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or anucleotide sequence having at least 60% identity thereto after optimalalignment, or capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 orSEQ ID NO:13 or SEQ ID NO:15 or a complementary form thereof under lowstringency conditions; (ix) SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16or SEQ ID NO:18 or a nucleotide sequence having at least 60% identitythereto after optimal alignment, or capable of hybridizing to SEQ IDNO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a complementaryform thereof under low stringency conditions; (x) SEQ ID NO:19 or SEQ IDNO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:27 ora nucleotide sequence having at least 60% identity thereto after optimalalignment, or capable of hybridizing to SEQ ID NO:19 or SEQ ID NO:21 orSEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:27 or acomplementary form thereof under low stringency conditions; (xi) SEQ IDNO:28 or SEQ ID NO:30 or a nucleotide sequence having at least 60%identity thereto after optimal alignment, or capable of hybridizing toSEQ ID NO:28 or SEQ ID NO:30 or a complementary form thereof under lowstringency conditions; (xii) SEQ ID NO:31 or SEQ ID NO:33 or anucleotide sequence having at least 60% identity thereto after optimalalignment, or capable of hybridizing to SEQ ID NO:31 or SEQ ID NO:33 ora complementary form thereof under low stringency conditions;
 58. Themethod of claim 56 wherein the bioproduct is p-hydroxybenzoic acid(pHBA) and the genetic sequence comprises one or more genetic sequencesselected from the list comprising a nucleotide sequence encodinghydroxycinnamoyl-CoA hydratase/lysase, a nucleotide sequence encodingchorismate pyruvate lyase, a nucleotide sequence encoding the ubiC genefrom E. coli, a nucleotide sequence encoding the HCHL gene fromPseudomonas fluorescens
 59. The method of claim 56 wherein thebioproduct is vanillin and the enzyme encoded by the genetic sequence isselected from the list comprising 3-dehydroshikimate dehyhratase,catechol-O-methyltransferase, aryl aldehyde dehybrogenase, feruloyl-CoAsynthetase, enoyl-CoA hydratase and enoyl-CoA aldolase.
 60. The methodof claim 56 wherein the bioproduct is sorbitol and the enzyme encoded bythe genetic sequence is glucose-fructose oxidoreductase.
 61. The methodof claim 60 wherein the glucose-fructose oxidoreductases is encoded bythe polynucleotide sequence set forth in GenBank Accession numberZ80356, or a homolog thereof having at least 60% identity thereto afteroptimal alignment, or capable of hybridizing to GenBank Accession numberZ80356 or a complementary form thereof under low stringency conditions.62. The method of claim 60 wherein the glucose-fructose oxidoreductaseis encoded by the polynucleotide sequence set forth in GenBank Accessionnumber M97379, or a homolog thereof having at least 60% identity theretoafter optimal alignment, or capable of hybridizing to GenBank Accessionnumber M97379 or a complementary form thereof under low stringencyconditions.
 63. The method of claim 56 wherein the bioproduct is fructanand the enzyme encoded by the genetic sequence is selected from the listcomprising fructosyltransferase and levan sucrase.
 64. The method ofclaim 63 wherein the fructosyltransferase is encoded by thepolynucleotide sequence set forth in GenBank Accession number AY150365,or a homolog thereof having at least 60% identity thereto after optimalalignment, or capable of hybridizing to GenBank Accession numberAY150365 or a complementary form thereof under low stringencyconditions.
 65. A vector comprising a genetic sequence encoding anenzyme of claim
 55. 66. The vector of claim 65 wherein the vector is anexpression vector.
 67. A genetically modified C4 grass cell or group ofcells comprising an introduced genetic sequence encoding an enzyme ofclaim
 55. 68. A genetically modified C4 grass cell or group of cellscomprising an introduced genetic sequence encoding a vector of claim 65.69. A genetically modified C4 grass plant comprising a cell or group ofcells of claim 67 or genetically modified progeny thereof.
 70. Seeds orother reproductive material from the plant of claim
 68. 71. A productproduced in a genetically modified plant or genetically modified cellsor parts of a plant by the method of claim 55.